Kinetic models of C3H mouse mammary tumor growth: implications regarding tumor cell loss

Three models of tumor cell loss are described. The effects of cell loss on other cellular kinetic parameters are evaluated, and experiments which may distinguish among the models are discussed. Each model is based on a different cell-loss mechanism, and equations for the cell-cycle, cell-frequency distribution, the growth of both the proliferating and non-proliferating cell population, the growth fraction (GF), and the relative rate of volumetric growth, (dV/dt)/V, are derived. The following types of data are simulated for each model: the pulse labelling index, the mitotic index, and the labeling index as a function of time after a single or a series of 3H-TdR injections. The relative volumetric growth rate has the same mathematical form for each model. The PLM curves predicted by each model for the tumor lines studied (S102F and Slow) are not appreciably different. The predicted initial labeling index and mitotic index may differ significantly among the models depending upon the tumor line. The most striking difference among the models lies in the predictions regarding the labeling index as a function of time after a single or after a series of 3H-TdR injections. These types of labeling experiments should be valuable for distinguishing the different cell-loss mechanisms in solid tumors.     

Kinetic Models of C3h Mouse Mammary Tumor Growth: Implications Regarding Tumor Cell Loss

Three models of tumor cell loss are described. the effects of cell loss on other cellular kinetic parameters are evaluated, and experiments which may distinguish among the models are discussed. Each model is based on a different cell-loss mechanism, and equations for the cell-cycle, cell-frequency distribution, the growth of both the proliferating and non-proliferating cell population, the growth fraction (GF), and the relative rate of volumetric growth, (dV/dt)/V, are derived. The following types of data are simulated for each model: the pulse labelling index, the mitotic index, and the labeling index as a function of time after a single or a series of ³H-TdR injections. the relative volumetric growth rate has the same mathematical form for each model. the PLM curves predicted by each model for the tumor lines studied (S102F and Slow) are not appreciably different. the predicted initial labeling index and mitotic index may differ significantly among the models depending upon the tumor line. the most striking difference among the models lies in the predictions regarding the labeling index as a function of time after a single or after a series of ³H-TdR injections. These types of labeling experiments should be valuable for distinguishing the different cell-loss mechanisms in solid tumors.     

shRNA-mediated silencing of Gli2 gene inhibits proliferation and sensitizes human hepatocellular carcinoma cells towards TRAIL-induced apoptosis

Aberrant activation of the Hedgehog (Hh) signaling pathway has been reported in various cancer types including hepatocellular carcinoma (HCC). As a key effector of this signaling, Gli2 plays a crucial role in carcinogenesis, including the activation of genes encoding apoptosis inhibitors and cell-cycle regulators. In this study, we examined the role of Gli2 proliferation and survival of HCC cells. First, the expression levels of Hh pathway components were detected in a subset of HCC cell lines. To establish the role of Gli2 in maintaining the tumorigenic properties of HCC cells, we developed small hairpin RNA (shRNA) targeting Gli2 and transfected it into SMMC-7721 cell, which was selected with high level of Hh signaling expression. Next, effects of Gli2 gene silencing, on cell proliferation and on the expression of cell cycle-related proteins were evaluated, then, whether down-regulation of Gli2 renders HCC cell susceptible to TRAIL was examined in vitro. Knockdown of Gli2 inhibited cell proliferation and induced G1 phase arrest of cell cycle in SMMC-7721 cell through down-regulation of cyclin D1, cyclinE2, and up-regulation of p21-WAF1. Also, Gli2 gene siliencing sensitized SMMC-7721 cell to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by reducing the expression of the long and short isoform of c-FLIP and Bcl-2, and then augmented the activation of initiator caspases-8/-9 and effector caspases-3, which induces PARP cleavage. In conclusion, our data suggest that Gli2 plays a predominant role in the proliferation and apoptosis resistance of HCC cells, and that knockdown of Gli2 may be a novel anticancer strategy for the treatment of HCC.     

Retracted: MicroRNA-34a inhibit hepatocellular carcinoma progression by repressing hexokinase-1

Hepatocellular carcinoma (HCC) is known as a frequent type of primary cancer in the liver, and it is the third-most common cause of cancer-related death all over the world. However, the molecular mechanism in the progression of HCC is still unclear. The current study was designed to investigate the expression and function of microRNA-34a (miR-34a) in HCC. In HCC tissues and cells, the expression levels of miR-34a were analyzed by quantitative real-time polymerase chain reaction. The association between the level of miR-34a and hexokinase (HK)-1 was also investigated via luciferase reporter assay. Cell viability and proliferation were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry. To assess whether miR-34a can limit tumor growth in vivo, animal models and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were used for examining the role of miR-34a on the development of HCC and cell apoptosis. The expression level of miR-34a was reduced in HCC samples and cells. The expression of miR-34a was associated with the viability and proliferation capacity of HCC cells, and miR-34a could inhibit HCC cells proliferation by inhibiting HK1. In the mouse model of HCC, volumes and weight of the tumors were significantly decreased by transfection with miR-34a mimic compared with the control group. Furthermore, miR-34a mimics could induce apoptosis in a greater proportion of cells compared with the control group. Taken together, the data may provide some novel insights into the molecular mechanism of miR-34a and HK1 in the progression of HCC. Thus, miR-34a/HK1 axis might be a novel promising therapeutic target for treating HCC.     

FT Raman spectroscopy in the evaluation of biomarkers of normal and pathological placenta tissue

Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumors with high recurrence and metastasis rates. Radiotherapy represents a major therapeutic option for HCC patients. However, the efficacy of radiotherapy has been limited due to the development of intrinsic radioresistance of the tumor cells. Small ubiquitin-like modifier 1 pseudogene 3 (SUMO1P3), one member of SUMO pseudogene family, is a novel identified lncRNA that was originally identified to be upregulated in gastric cancer. However, the detailed roles of SUMO1P3 in HCC development remain to be elucidated. Here, the expression of SUMO1P3 in HCC tissues and cells was examined by qRT-PCR. Cell proliferation, colony formation ability, invasion ability, apoptosis, and radiosensitivity were detected by MTT assay, colony formation assay, cell invasion assay, flow cytometry analysis, and survival fraction assay, respectively. We found that SUMO1P3 was significantly upregulated in HCC tissues and cells. Besides, SUMO1P3 was highly expressed in HCC patients with higher TNM stage. Furthermore, SUMO1P3 knockdown markedly suppressed cell proliferation, colony formation ability, and cell invasiveness, promoted apoptosis, and enhanced radiosensitivity of HCC cells. We concluded that the knockdown of SUMO1P3 repressed tumor growth, invasion, promoted apoptosis, and enhanced radiosensitivity in HCC, providing evidence that SUMO1P3 might be a potential novel biomarker and a therapeutic target for HCC.

     

Salinomycin Inhibits Proliferation and Induces Apoptosis of Human Hepatocellular Carcinoma Cells In Vitro and In Vivo

The anti-tumor antibiotic salinomycin (Sal) was recently identified as a selective inhibitor of breast cancer stem cells; however, the effect of Sal on hepatocellular carcinoma (HCC) is not clear. This study aimed to determine the anti-tumor efficacy and mechanism of Sal on HCC. HCC cell lines (HepG2, SMMC-7721, and BEL-7402) were treated with Sal. Cell doubling time was determinated by drawing growth curve, cell viability was evaluated using the Cell Counting Kit 8. The fraction of CD133⁺ cell subpopulations was assessed by flow cytometry. We found that Sal inhibits proliferation and decreases PCNA levels as well as the proportion of HCC CD133⁺cell subpopulations in HCC cells. Cell cycle was analyzed using flow cytometry and showed that Sal caused cell cycle arrest of the various HCC cell lines in different phases. Cell apoptosis was evaluated using flow cytometry and Hoechst 33342 staining. Sal induced apoptosis as characterized by an increase in the Bax/Bcl-2 ratio. Several signaling pathways were selected for further mechanistic analyses using real time-PCR and Western blot assays. Compared to control, β-catenin expression is significantly down-regulated upon Sal addition. The Ca²⁺ concentration in HCC cells was examined by flow cytometry and higher Ca²⁺ concentrations were observed in Sal treatment groups. The anti-tumor effect of Sal was further verified in vivo using the hepatoma orthotopic tumor model and the data obtained showed that the size of liver tumors in Sal-treated groups decreased compared to controls. Immunohistochemistry and TUNEL staining also demonstrated that Sal inhibits proliferation and induces apoptosis in vivo. Finally, the role of Sal on in vivo Wnt/β-catenin signaling was evaluated by Western blot and immunohistochemistry. This study demonstrates Sal inhibits proliferation and induces apoptosis of HCC cells in vitro and in vivo and one potential mechanism is inhibition of Wnt/β-catenin signaling via increased intracellular Ca²⁺ levels.     

肿瘤抑素抗肿瘤相关肽对肝癌细胞增殖和凋亡的影响

肿瘤抑素抗肿瘤相关肽-19肽是由肿瘤抑素185~203位氨基酸组成, 具有直接抑制黑色素瘤细胞生长作用, 但其对肝癌细胞增殖和凋亡是否有影响, 对肝癌是否具有治疗作用还需进一步研究。本研究中采用基因工程技术将合成19肽基因与载体pTYB2重组后进行蛋白表达、纯化获得19肽。通过MTT法、生长曲线观察19肽对人肝癌细胞生长抑制作用; TUNEL标记法、流式细胞仪细胞周期检测法、透射电镜观察19肽对肝癌细胞凋亡的影响; 小鼠H22腹水型转移型肝癌实体瘤抑瘤实验证明其体内的抑瘤作用。MTT实验和生长曲线实验表明随着19肽浓度的增加肝癌细胞的存活率下降。在相同19肽浓度下, 随着作用时间延长存活细胞逐渐减少。电镜观察治疗组细胞出现明显凋亡, 流式细胞仪可检测到前G1峰, TUNEL标记法也证实治疗组可见明显的凋亡细胞, 体内19肽作用的小鼠H22腹水型转移型肝癌的抑瘤率达48.46%。可见, 肿瘤抑素19肽可抑制肝癌细胞生长, 促进肝癌细胞凋亡, 对肝癌具有一定的治疗作用。     

microRNA-802 accelerates hepatocellular carcinoma growth by targeting RUNX3

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Prognosis is often unfavorable. In this study, the effects of microRNA-802 (miR-802) on HCC progression were assessed in vivo and in vitro. miR-802 was found to be significantly upregulated in HCC tumor tissue compared to paired adjacent nontumor tissue. In vitro, transfection with a miR-802 mimic accelerated SMMC-7721 cellular proliferation, increased accumulation of the cell-cycle S-phase cell populations, as well as cell migration. In vivo injection of a miR-802 agomir promoted HCC proliferation in nude mice. Targets of miR-802 were predicted by miRWalk, miRanda, RNA22, and Targetscan. By luciferase reporter assay RUNX3 was identified as a direct target of miR-802. As judged by western blot analysis, RUNX3 was upregulated when miR-802 was inhibited. These data demonstrate increased miR-802 expression in patients with HCC and that miR-802 overexpression promotes tumor cell growth, in a RUNX3-dependent manner.     

Small interfering RNA targeting integrin-linked kinase inhibited the growth and induced apoptosis in human bladder cancer cells

Integrin-linked kinase (ILK), an intracellular serine/threonine kinase, is implicated in cell growth and survival, cell-cycle progression, tumor angiogenesis, and cell apoptosis. Recent studies showed that the expression and activity of ILK increased significantly in many types of solid tumors. However, the exact molecular mechanism of ILK underlie tumor has not been fully ascertained. The purpose of our study was to determine whether knockdown of ILK would inhibit cell growth and induce apoptosis in bladder cancer cells using a plasmid vector based small interfering RNA (siRNA). The experiments showed that knockdown of ILK could remarkably inhibit cell proliferation and growth, regulate cell cycle and induce apoptosis of bladder cancer BIU-87 and EJ cells. We demonstrated that knockdown of ILK inhibited phosphorylation of downstream signaling targets protein kinase B/Akt, glycogen synthase kinase 3-beta (GSK-3β), and reduced expression of β-catenin in BIU-87 as well as EJ cells by Western blot and Immunofluorescence analysis. In addition, down-regulation of ILK also could increase expression of Ribonuclease inhibitor (RI), an important acidic cytoplasmic protein with many functions. BALB/C nude mice injected with the BIU-87 cells transfected ILK siRNA showed a significant inhibition of the tumor growth with lighter tumor weight, lower microvessels density and higher apoptosis rate than those in the other two control groups. In conclusion, these results suggest that ILK might be involved in the development of bladder cancer, and could be served as a novel potential therapy target for human bladder cancer. Our study may be of biological and clinical importance.     

Transmembrane protein 106C promotes the development of hepatocellular carcinoma

Several protein-coding genes have been identified to play essential roles in cancer biology, and they are dysregulated in many tumors. Transmembrane protein 106C (TMEM106C) is differentially expressed in several human and porcine diseases; however, the expression and biological functions of TMEM106C in hepatocellular carcinoma (HCC) are not clear. In our study, we obtained paired tissue samples from patients undergoing resection for HCC and public databases, which were analyzed for TMEM106C expression using quantitative real-time polymerase chain reaction (qRT-PCR). We further conducted in vitro and in vivo experiments in HCC cell lines and nude mice, respectively, in which TMEM106C was overexpressed or knocked down. Cell-Counting Kit-8 and colony formation experiments were used to determine the influence of TMEM106C on cell proliferation, flow cytometric assays were used to detect the influence on cell cycle distribution and apoptosis, and transwell assays were used for detecting changes in cell migration and invasion. TMEM106C levels were significantly elevated in HCC tissues and cell lines from public databases and our collected specimens from patients. Moreover, higher TMEM106C expression levels predicted a poor prognosis in HCC patients in survival analysis. Overexpression of TMEM106C in HCC cells accelerated cell growth, migration, and invasion, but it inhibited cell apoptosis by targeting forkhead box O-1 (FOXO1) and FOXO3. Conversely, TMEM106C knockdown impeded cell proliferation and metastasis, whereas it enhanced the rate of apoptosis. More important, knockdown of the expression of TMEM106C in HCC cells inhibited the growth of xenograft tumors in vivo. Collectively, these results suggest that TMEM106C acts as an oncogene and can serve as a potential therapeutic target for HCC in the future.     

Visualizing the cell-cycle progression of endothelial cells in zebrafish

The formation of vascular structures requires precisely controlled proliferation of endothelial cells (ECs), which occurs through strict regulation of the cell cycle. However, the mechanism by which EC proliferation is coordinated during vascular formation remains largely unknown, since a method of analyzing cell-cycle progression of ECs in living animals has been lacking. Thus, we devised a novel system allowing the cell-cycle progression of ECs to be visualized in vivo. To achieve this aim, we generated a transgenic zebrafish line that expresses zFucci (zebrafish fluorescent ubiquitination-based cell cycle indicator) specifically in ECs (an EC-zFucci Tg line). We first assessed whether this system works by labeling the S phase ECs with EdU, then performing time-lapse imaging analyses and, finally, examining the effects of cell-cycle inhibitors. Employing the EC-zFucci Tg line, we analyzed the cell-cycle progression of ECs during vascular development in different regions and at different time points and found that ECs proliferate actively in the developing vasculature. The proliferation of ECs also contributes to the elongation of newly formed blood vessels. While ECs divide during elongation in intersegmental vessels, ECs proliferate in the primordial hindbrain channel to serve as an EC reservoir and migrate into basilar and central arteries, thereby contributing to new blood vessel formation. Furthermore, while EC proliferation is not essential for the formation of the basic framework structures of intersegmental and caudal vessels, it appears to be required for full maturation of these vessels. In addition, venous ECs mainly proliferate in the late stage of vascular development, whereas arterial ECs become quiescent at this stage. Thus, we anticipate that the EC-zFucci Tg line can serve as a tool for detailed studies of the proliferation of ECs in various forms of vascular development in vivo.     

Mathematical models of hierarchically structured cell populations under equilibrium with application to the epidermis

Abstract. There are three categories of keratinocytes in the germinative compartment of the epidermis – stem, transit-amplifying and post-mitotic. Their population structure is hierarchical. This means that stem cells differentiate into transit-amplifying cells which, after a few rounds of division, become post-mitotic cells. The cell processes of birth, differentiation, death and migration affect the composition and proliferation rate of the germinative compartment. These phenomena are quantified by various cell kinetic parameters. In this paper we derive equations that relate these parameters for different models of hierarchically structured cell populations in equilibrium. We include in the models asymmetric and symmetric division, variations in cell-cycle times, apoptosis and variation in the number of transit generations. We conclude that variation in cell-cycle times need only be considered if apoptosis is not negligible. If it is negligible, then only average cell-cycle times are needed. Unfortunately, it is impossible to predict the importance of apoptosis from the available experimental data. However, the strength of its effect is determined by the other parameters, especially the fraction of cycling stem cells. We show that variation in the number of transit generations can have a potentially large effect on cell birth rate. We also show that cell birth rate does not directly depend on the mean transit-amplifying cell-cycle time, only on the mean stem cell-cycle time. We argue that 'homogeneous cell population' equations should not be used to study hierarchical cell populations as has been done in the past. Finally we argue that stem cell parameters and transit-amplifying cell parameters should not be lumped together.     

shRNA interference for extracellular signal-regulated kinase 2 can inhibit the growth of esophageal cancer cell line Eca109

Background: Esophageal squamous cell carcinoma is one of the most common digestive tract cancers with 5-year survival rate less than 10% owing to its poor prognosis. Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway has been mainly involved in the pathogenesis of various cancers. In present study, we investigated the role of ERK2 in human esophageal cancer cell line Eca109.

Methods: Short-hairpin RNA (shRNA) interference vector targeted ERK2 was constructed using pGeneclip U1 hairpin cloning systems, then transfected into Eca109 cell line. The transfection efficiency was observed by fluorescence microscope and cell growth after transfection with shRNA-ERK2 vector was determined by methylthiazolyl blue tetrazolium (MTT) assay. The ERK2 expression after transfection was detected by western-blotting. The cell apoptosis and cell-cycle was analyzed by flow cytometry. The role of p-ERK2 was confirmed by immunohistochemistry and soft agar colony formation assay.

Results: The growth of Eca109 transfected with shRNA-ERK2 vector was obviously inhibited compared to control group via MTT analysis. The inhibition rate after transfection with shRNA-ERK2 for 96?h was 10.45%, the expression of ERK2 was obviously reduced compared to the control analyzed by western-blot, cell apoptosis was 9.7% (compared to control, P?<?0.05), and cell-cycle was arrested at G1 phase.

Conclusions: In present study we demonstrated for the first time that transfection with shRNA-ERK2 targeted ERK2 into Eca109 cells can inhibit growth of Eca109, inducing cell apoptosis and influencing cell-cycle. Together, these results we obtained suggested that ERK2 plays an important role in cell growth of Eca109.     

Fully automated TV-image analysis of the cell-cycle: comparison of the PLM method with determinations of the percentage and the DNA content of labelled cells

A cell-cycle analysis based on a fully automated TV-image scanning system is proposed to replace the laborious PLM method. To compare the efficiency of the two procedures, cell-cycle parameters were assessed in Ehrlich (diploid and hyperdiploid), L-1210, and JB-1 mouse ascites tumours and in rat jejunal crypts. The percentages of labelled mitoses (PLM) were counted visually on Feulgen-stained autoradiographs obtained at various times after a single 3H-thymidine pulse. The fraction of labelled cells (P) and the DNA ratio of labelled and unlabelled cells were measured by TV-image analysis in the same slides and plotted against time. Within practical limits, TV-image analysis using the P-curve gives the same results as the PLM method. Using the P-curve has the important advantage that its first part, beginning at the time of 3H-thymidine injection and ending at the first maximum, furnishes more information about the cell cycle than the corresponding part of the PLM curve. It can be used to compute tG2M tS and the ratio of the growth faction index to the cell-cycle time (IP/tC) whereas the first part of the PLM-curve reveals only the length of the S-phase (tS). The IP/tC ratio is a readily accessible measure of growth and increases when the cells divide more frequently. Cell death rates may be neglected since the ratio is determined within less than the duration of one cell cycle. Moreover, the data from the first part of the P curve indicate whether there is a large non-growth fraction. If the non-growth fraction is small, i.e. if IP approximately 1, the P curve need only be measured until the first maximum is reached so that fewer samples and animals are required. If the non-growth fraction is large or unknown, the cell-cycle parameters are calculated by reference to the position and size not only of the first minimum and the first maximum, but also of the second minimum of the P curve.     

MicroRNA-450b-3p inhibits cell growth by targeting phosphoglycerate kinase 1 in hepatocellular carcinoma

Dysregulation of microRNAs frequently contributes to the occurrence and progression of human diseases, including hepatocellular carcinoma (HCC). In this study, the role of miR-450b-3p in HCC was investigated. Gene Expression Omnibus database and HCC specimens were used to evaluate the expression level of miR-450b-3p and the patient's prognosis. Cell functional analyses and tumor xenograft model were used to assess the role of miR-450b-3p in HCC. Bioinformatics was used to predict the downstream target gene of miR-450b-3p, which was verified by dual-luciferase reporter assay. MiR-450b-3p was found to be downregulated in HCC cell lines and tissues, compared with nontransformed immortal hepatic cells and adjacent normal liver tissues, respectively. Lower expression of miR-450b-3p was associated with poor overall survival and disease-free survival in patients with HCC. Ectopic expression of miR-450b-3p inhibited HCC cell viability, colony formation, and cell-cycle progression in vitro, and suppressed the growth of HCC xenograft tumors in vivo. Interestingly, a negative correlation between miR-450b-3p and phosphoglycerate kinase 1 (PGK1) protein was observed among HCC specimens. Additionally, miR-450b-3p inhibited PGK1 expression and phosphorylation of protein kinase B in HCC cell lines. Further experiments confirmed that PGK1 was a direct target of miR-450b-3p. Moreover, restoration of PGK1 abrogated the inhibitory effect of miR-450b-3p on HCC proliferation and cell division. In conclusion, miR-450b-3p is downregulated in human HCC and exerts tumor suppressive effects at least in part by inhibiting PGK1.     

Role of TNF-alpha in gut mucosal changes after severe burn

Gut epithelial cell death by apoptosis is increased in the gut epithelium after severe burn associated with mucosal atrophy. We hypothesized that tumor necrosis factor (TNF)-alpha-TNF receptor (TNFR) interaction activates apoptosis in small bowel mucosal cells after severe burn. C57BL6 mice received a 30% total body surface area scald burn and were treated with neutralizing anti-TNF-alpha. The proximal small bowel was assessed for mucosal atrophy. Proliferation and apoptosis of mucosal cells were assessed by proliferative cell nuclear antigen-immunostaining and terminal deoxyuridine nick-end labeling assay, respectively. Mucosal height and mucosal cell number decreased after burn. Anti-TNF-alpha-treated mice showed significantly less mucosal atrophy. Proliferation of intestinal cells was not changed with burn or anti-TNF-alpha treatment. An over threefold increase in apoptotic cell number was seen after burn, which was diminished by anti-TNF-alpha treatment. Changes in gut mucosal homeostasis after severe burn are affected, in part, by the activation of apoptosis by TNF-alpha-TNFR interaction.