Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

Ultraviolet A (UVA) radiation (λ = 320–400 nm) is considered a major cause of human skin cancer. Pin1, a peptidyl prolyl isomerase, is overexpressed in most types of cancer tissues and plays an important role in cell proliferation and transformation. Here, we demonstrated that Pin1 expression was enhanced by low energy UVA (300–900 mJ/cm²) irradiation in both skin tissues of hairless mice and JB6 C141 epidermal cells. Exposure of epidermal cells to UVA radiation increased cell proliferation and cyclin D1 expression, and these changes were blocked by Pin1 inhibition. UVA irradiation also increased activator protein-1 (AP-1) minimal reporter activity and nuclear levels of c-Jun, but not c-Fos, in a Pin1-dependent manner. The increases in Pin1 expression and in AP-1 reporter activity in response to UVA were abolished by N-acetylcysteine (NAC) treatment. Finally, we found that pre-exposure of JB6 C141 cells to UVA potentiated EGF-inducible, anchorage-independent growth, and this effect was significantly suppressed by Pin1inhibition or by NAC.     

Antizyme1基因转染对K562细胞增殖与凋亡的影响

研究鸟氨酸脱羧酶抗酶蛋白对人红白血病K562细胞增殖、三氧化二砷（ As2O3）诱导凋亡时的影响。方法: 定点突变技术构建缺失frameshift位点的pEGFP-N1-AZ1-mutation重组表达载体。脂质体法转染K562细胞,通过G418筛选获得稳定表达antizyme1的K562pAZ1m细胞系。采用不同浓度的As2O3处理细胞,通过MTT法检测细胞增殖,流式细胞术分析细胞周期及凋亡变化。并通过RT-PCR方法检测antiyme1转染对cyclin D1和survivin基因表达的影响。结果：获得稳定表达antizyme1的K562-AZ1m细胞株后,其增殖能力明显减慢。CyclinD1基因表达降低,细胞主要停滞于G0/G1期。在 As2O3的诱导作用下,细胞凋亡增多,survivin基因表达降低。结论：AZ1基因能够抑制K562细胞增殖,通过对cyclinD1的负调控使细胞周期停滞于G0/G1期。并可能通过下调survivin表达来加强 As2O3对其的诱导凋亡作用     

Role of SOCE architects STIM and Orai proteins in Cell Death

Calcium (Ca²⁺) signaling plays a critical role in regulating plethora of cellular functions including cell survival, proliferation and migration. The perturbations in cellular Ca²⁺ homeostasis can lead to cell death either by activating autophagic pathways or through induction of apoptosis. Endoplasmic reticulum (ER) is the major storehouse of Ca²⁺ within cells and a number of physiological agonists mediate ER Ca²⁺ release by activating IP₃ receptors (IP₃R). This decrease in ER Ca²⁺ levels is sensed by STIM, which physically interacts and activates plasma membrane Ca²⁺ selective Orai channels. Emerging literature implicates a key role for STIM1, STIM2, Orai1 and Orai3 in regulating both cell survival and death pathways. In this review, we will retrospect the work highlighting the role of STIM and Orai homologs in regulating cell death signaling. We will further discuss the rationales that could explain the dual role of STIM and Orai proteins in regulating cell fate decisions.     

Sinensetin isolated from Orthosiphon aristatus inhibits cell proliferation and induces apoptosis in hepatocellular carcinoma cells

Orthosiphon aristatus is a traditional folk medicine extensively used in Southeast Asia because of its various pharmacological effects, including antioxidant, antitumor, and hypoglycemic activities. Orthosiphon extracts have been found to be cytotoxic to hepatocellular carcinoma (HCC) cells, which is attributed to their phytochemical content. However, the mechanism of action underlying the cytotoxic effects remains unclear. Hence, the present study investigated the effect of Sinensetin purified from O. aristatus on HCC in vitro. Sinensetin was isolated from O. aristatus leaves and the chemical structure was confirmed by ultra violet (UV)-vis, infrared (IR), nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS). The results revealed that 24-h treatment with the purified compound markedly inhibited the survival of HepG2 cells, with IC₅₀ of 39.93 ± 1.10 μg/mL. HepG2 cells treated with the IC₅₀ of Sinensetin showed characteristic morphological changes, as determined by PI and AO/Etbr dual staining, including DNA fragmentation, thus confirming the apoptosis induction. Sinensetin induced cell cycle arrest at G0/G1 phase, and the data were substantiated by flow cytometry. Furthermore, Sinensetin modulated key signaling molecules; anti-apoptotic Bcl-xL was down-regulated, whereas the expressions of tumor suppressors TRAIL and PTEN were up-regulated. We conclude that Sinensetin can be effective against HCC.     

高温对乳腺上皮细胞生长及凋亡的影响

近年来随着生物技术的发展,对应激的研究已经深入到细胞水平上,Li met al·(2006)揭示细胞抵抗热应激的能力因细胞种类、热处理的温度、时间的不同而存在差异;热应激还会引起G1/S或G2/M细胞周期调控点的阻滞(Kuhl and Rensing,2002;Fuse et al.,1996),剧烈的热应激还可以诱导细     

Cyclic mechanical stretch contributes to network development of osteocyte-like cells with morphological change and autophagy promotion but without preferential cell alignment in rat

Osteocytes play important roles in controlling bone quality as well as preferential alignment of biological apatite c-axis/collagen fibers. However, the relationship between osteocytes and mechanical stress remains unclear due to the difficulty of three-dimensional (3D) culture of osteocytes in vitro. The aim of this study was to investigate the effect of cyclic mechanical stretch on 3D-cultured osteocyte-like cells. Osteocyte-like cells were established using rat calvarial osteoblasts cultured in a 3D culture system. Cyclic mechanical stretch (8% amplitude at a rate of 2 cycles min^?1) was applied for 24, 48 and 96 consecutive hours. Morphology, cell number and preferential cell alignment were evaluated. Apoptosis- and autophagy-related gene expression levels were measured using quantitative PCR. 3D-cultured osteoblasts became osteocyte-like cells that expressed osteocyte-specific genes such as Dmp1, Cx43, Sost, Fgf23 and RANKL, with morphological changes similar to osteocytes. Cell number was significantly decreased in a time-dependent manner under non-loaded conditions, whereas cyclic mechanical stretch significantly prevented decreased cell numbers with increased expression of anti-apoptosis-related genes. Moreover, cyclic mechanical stretch significantly decreased cell size and ellipticity with increased expression of autophagy-related genes, LC3b and atg7. Interestingly, preferential cell alignment did not occur, irrespective of mechanical stretch. These findings suggest that an anti-apoptotic effect contributes to network development of osteocyte-like cells under loaded condition. Spherical change of osteocyte-like cells induced by mechanical stretch may be associated with autophagy upregulation. Preferential alignment of osteocytes induced by mechanical load in vivo may be partially predetermined before osteoblasts differentiate into osteocytes and embed into bone matrix.     

自噬在疾病中的双重作用

自噬是细胞的一种正常的生理活动,参与细胞内损伤的蛋白质和亚细胞器经溶酶体途径降解的过程。自噬可以抵御外界的不良环境,在多种疾病中起着重要作用。近年来,大量研究表明自噬在细胞新陈代谢和生理功能上有双重作用,在疾病发生的不同时期,自噬起到不同的作用。通常情况自噬可以及时的清除细胞内损伤的蛋白质,作为一种细胞的保护机制,但是自噬的持续活化,导致细胞内大量蛋白质的降解,使细胞无法维持其基本结构,最终将导致细胞坏死或凋亡。自噬、凋亡和坏死的转化,很有可能受到p53、Bcl-2、Beclin-1、ATG5、TG2及p62等信号分子调控。肝脏和心脏是维持人体生命活动的重要器官,自噬在脂肪肝、肝硬化、心肌梗塞及心脏衰竭等疾病中扮演着重要的角色。本文总结了自噬、凋亡及坏死的相互关系,自噬在疾病中的双重作用,并重点介绍自噬在肝脏和心脏疾病中的作用。     

VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.     

Adenovirus vector-mediated FAM176A overexpression induces cell death in human H1299 non-small cell lung cancer cells

FAM176A (family with sequence similarity 176 member A) is a novel molecule related to programmed cell death. A decreased expression of FAM176A has been found in several types of human tumors in including lung cancers. In the present study, we investigated the biological activities of FAM176A on the human non–small cell lung cancer cell line H1299 cells. We constructed a recombinant adenovirus 5-FAM176A vector (Ad5-FAM176A) and evaluated the expression and anti-tumor activities in vitro. Cell viability analysis revealed that the adenovirus-mediated increase of FAM176A inhibited the growth of the tumor cells in a dose- and time-dependent manner. This inhibitory effect was mediated by both autophagy and apoptosis that involved caspase activation. In addition, cell cycle analysis suggested that Ad5-FAM176A could induce cell cycle arrest at the G2/M phase, all of which suggested that adenovirus-mediated FAM176A gene transfer might present a new therapeutic approach for lung cancer treatment. [BMB Reports 2014; 47(2): 104-109]     

A natural phenylpropionate derivative from Mirabilis himalaica inhibits cell proliferation and induces apoptosis in HepG2 cells

Bioactivity-guided study led to the isolation of a natural phenylpropionate derivative, (E)-3-(4-hydroxy-2-methoxyphenyl)-propenoic acid 4-hydroxy-3-methoxyphenyl ester from the roots of Mirabilis himalaica. Cellular analysis showed that compound 1 specifically inhibited the cancer cell growth through the S phase arrest. Mechanistically, compound 1 was able to induce the apoptosis in HepG2 cells through mitochondrial apoptosis pathway in which Bcl-2 and p53 were required. Interestingly, the cellular phenotype of compound 1 were shown specifically in cancer cells originated from hepatocellular carcinoma (HepG2) while compromised influence by compound 1 were detected within the normal human liver cells (L-02). Consistently, the in vivo inhibitory effects of compound 1 on tumor growth were validated by the in xenograft administrated with HepG2 cells. Our results provided a novel compound which might serve as a promising candidate and shed light on the therapy of the hepatocellular carcinoma.     

Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies

Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.     

Autophagy in mammalian cells

Autophagy is a regulated process for the degradation of cellular components that has been well conserved in eukaryotic cells. The discovery of autophagy-regulating proteins in yeast has been important in understanding this process. Although many parallels exist between fungi and mammals in the regulation and execution of autophagy, there are some important differences. The preautophagosomal structure found in yeast has not been identified in mammals, and it seems that there may be multiple origins for autophagosomes, including endoplasmic reticulum, plasma membrane and mitochondrial outer membrane. The maturation of the phagophore is largely dependent on 5’-AMP activated protein kinase and other factors that lead to the dephosphorylation of mammalian target of rapamycin. Once the process is initiated, the mammalian phagophore elongates and matures into an autophagosome by processes that are similar to those in yeast. Cargo selection is dependent on the ubiquitin conjugation of protein aggregates and organelles and recognition of these conjugates by autophagosomal receptors. Lysosomal degradation of cargo produces metabolites that can be recycled during stress. Autophagy is an impor-tant cellular safeguard during starvation in all eukaryotes; however, it may have more complicated, tissue specific roles in mammals. With certain exceptions, autophagy seems to be cytoprotective, and defects in the process have been associated with human disease.     

TRPs and Ca2+ in cell death and survival

Transient Receptor Potential (TRP) family mediate the influx of monovalent and/or divalent cations into cells in response to environmental stimuli. Pharmacological or genetic manipulations of TRP channels demonstrate that TRP channels influence cell death rates, prolonging or shortening of cell survival. Due to their diverse cellular localization, TRP channels mediated Ca²⁺ influx generates distinct intracellular Ca²⁺ signals that regulate downstream pathways converging to apoptosis or survival. In this review, we summarize the accumulated knowledge focused on how TRP channel regulate cell fate and may affect different pathologies including cardiovascular, neurological, metabolic or neoplastic disorders.     

Lysosomes and lysosomal cathepsins in cell death

Lysosomes are the key degradative compartments of the cell. Lysosomal cathepsins, which are enclosed in the lysosomes, help to maintain the homeostasis of the cell's metabolism by participating in the degradation of heterophagic and autophagic material. Following the targeted lysosomal membrane's destabilization, the cathepsins can be released into the cytosol and initiate the lysosomal pathway of apoptosis through the cleavage of Bid and the degradation of the anti-apoptotic Bcl-2 homologues. Cathepsins can also amplify the apoptotic signaling, when the lysosomal membranes are destabilized at a later stage of apoptosis, initiated by other stimuli. However, the functional integrity of the lysosomal compartment during apoptosis enables efficient autophagy, which can counteract apoptosis by providing the energy source and by disposing the damaged mitochondria, which generate the ROS. Impairing autophagy by disabling the lysosome function is being investigated as an adjuvant therapeutic approach to sensitize cells to apoptosis-inducing agents. Destabilization of the lysosomal membranes by the lysosomotropic detergents seems to be a promising strategy in this context as it would not only disable autophagy, but also promote apoptosis through the initiation of the lysosomal pathway. In contrast, the impaired autophagy and lysosomal degradation linked with the increased oxidative stress underlie degenerative changes in the aging neurons. This further suggests that lysosomes and lysosomal cathepsins have a dual role in cell death. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Role of Dicer1 in thyroid cell proliferation and differentiation

DICER1 plays a central role in the biogenesis of microRNAs and it is important for normal development. Altered microRNA expression and DICER1 dysregulation have been described in several types of tumors, including thyroid carcinomas. Recently, our group identified a new somatic mutation (c.5438A>G; E1813G) within DICER1 gene of an unknown function. Herein, we show that DICER1 is overexpressed, at mRNA level, in a significant-relative number of papillary (70%) and anaplastic (42%) thyroid carcinoma samples, whereas is drastically downregulated in all the analyzed human thyroid carcinoma cell lines (TPC-1, BCPAP, FRO and 8505c) in comparison with normal thyroid tissue samples. Conversely, DICER1 is downregulated, at protein level, in PTC in comparison with normal thyroid tissues. Our data also reveals that DICER1 overexpression positively regulates thyroid cell proliferation, whereas its silencing impairs thyroid cell differentiation. The expression of DICER1 gene mutation (c.5438A>G; E1813G) negatively affects the microRNA machinery and cell proliferation as well as upregulates DICER1 protein levels of thyroid cells but has no impact on thyroid differentiation. In conclusion, DICER1 protein is downregulated in papillary thyroid carcinomas and affects thyroid proliferation and differentiation, while DICER1 gene mutation (c.5438A>G; E1813G) compromises the DICER1 wild-type-mediated microRNA processing and cell proliferation.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

Influence of chk1 and plk1 silencing on radiation- or cisplatin-induced cytotoxicity in human malignant cells

The G2/M checkpoint is an attractive pathway for targeting and sensitizing tumor cells to cancer treatment. Abrogation of the G2/M checkpoint by targeting molecules, such as checkpoint kinase 1 (chk1), increases DNA breakage and sensitizes tumor cells to anti-tumoral agents. However, most of the previously described G2/M abrogators are actually targeting the G2-M border checkpoints rather than mitotic checkpoints. This prompted us to test the effects of combined targeting of chk1 and a critical regulator of mitosis, polo-like kinase 1 (plk1). Chk1 and plk1 were found to be co-expressed in 70% of primary neoplastic tissues we examined. Asynchronized tumor cells were treated with different DNA damaging-agents to activate G1/S, S or G2/M checkpoints. Either chk1 or plk1-specific antisense oligodeoxynucleotides (ASODN) enhanced DNA damaging agent-induced apoptosis. When used in combination, however, chk1- plus plk1-specific ASODN failed to produce synergistic effects. Moreover, selective targeting of plk1 or chk1 in tumor xenografts of mice by oncolytic adenovirus mutants demonstrated potent anti-tumoral efficacy in the presence of low dose cisplatin. Again, combined targeting of chk1 and plk1 did not further enhance anti-tumoral efficacy. We concluded that combined targeting of chk1 and plk1 was not superior to either targeting chk1 or plk1 alone, which suggested that chk1 and plk1 silencing might overlap in their mechanism of action. Whether combined targeting of chk1 with other, more specific mitotic regulators would synergistically sensitize tumor to anti-neoplastic therapeutics needs to be further clarified. Qinglei Gao and Xiaoyuan Huang contributed equally to this work.     

Role of epidermal growth factor-stimulated protein kinase in control of proliferation of A431 cells

Epidermal growth factor (EGF), which stimulates tyrosine-specific protein kinase activity both in vivo and in vitro, inhibits proliferation of A431 human epidermoid carcinoma cells. After mutagenesis clonal cell lines that were resistant to the growth inhibitory effects of EGF were selected. All six variants examined contained decreased EGF-stimulated protein kinase. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in variant cell lines with up to tenfold reductions in both activities. This result suggests that both EGF binding and kinase activities reside in the same or closely coupled molecules. The effect of EGF on growth of two resistant variants was examined in detail. Clone 29 contains approximately 50% and clone 4 contains approximately 20% of the EGF-stimulated protein kinase activity of the parental A431 cell line. In serum-supplemented medium, EGF stimulated proliferation of clone 29 but did not affect growth of clone 4. In a 1:1 mixture of DME and F-12 medium without serum, EGF caused both clone 29 and clone 4 to grow as well as in 10% serum. These variants, which were selected for resistance to the growth inhibitory effects of EGF, thus exhibit a strong mitogenic response to EGF. This result suggests that resistance to the growth inhibitory effect of EGF may involve both a decrease in EGF-stimulated protein kinase and an alteration in the response pathway.