Epidermal growth factor (EGF) induces apoptosis in a transfected cell line expressing EGF receptor on its membrane

Interaction between epidermal growth factor (EGF) and EGF receptor (EGFR) promotes cell growth in most cell lines, but in a number of cell lines, EGF paradoxically inhibits proliferation. In the present study, we established a cell line expressing full-length human EGFR on membrane with a GFP fluorescence reporter at the C-terminal and studied the effects of EGF on cell proliferation in the transfected cell line. Our results suggested that low concentrations of EGF promoted proliferation, while high concentrations of EGF induced loss of adhesion, cell cycle arrest, apoptosis, and inhibition of proliferation. The effects of EGF on cell proliferation correlated well with the expression levels of EGFR. High concentrations of EGF induced both EGFR expression and apoptosis in a dose-dependent manner. Our study reported, for the first time, a relationship between the effects of EGF on cell proliferation and levels of EGFR expression in one cell line expressing different levels of EGFR caused by different concentrations of EGF treatment. The study should provide considerable insight into the effects of EGF on cell proliferation and tumor cell metastasis.     

Estrogen receptor alpha negative breast cancer patients: estrogen receptor beta as a therapeutic target

Clinical management of breast cancer is increasingly guided by assessment of tumor phenotypic parameters. One of these is estrogen receptor (ER) status, currently defined by ERalpha expression. However with the discovery of a second ER, ERbeta and its variant isoforms, the definition of ER status is potentially more complex. In breast tumors there are two ERbeta expression cohorts. One where ERbeta is co-expressed with ERalpha and the other expressing ERbeta alone. In the latter subgroup of currently defined ER negative patients ERbeta has the potential to be a therapeutic target. Characterization of the nature and role of ERbeta in ERalpha negative tumors is essentially unexplored but available data suggest that the role of ERbeta may be different when co-expressed with ERalpha and when expressed alone. This review summarizes available data and explores the possibility that ERbeta signaling may be a therapeutic target in these tumors. Evidence so far supports the idea that the role of ERbeta in breast cancer is different in ERalpha negative compared to ERalpha positive tumors. However, cohort size and numbers of independent studies are small to date, and more studies are needed with better standardization of antibodies and protocols. Also, the ability to determine the role of ERbeta in ERalpha negative breast cancer and therefore assess ERbeta signaling pathways as therapeutic targets would be greatly facilitated by identification of specific downstream markers of ERbeta activity in breast cancer.     

The role of Bcl-2 and its combined effect with p21<Superscript>CIP1</Superscript> in adaptation of CHO cells to suspension and protein-free culture

The overexpression of the antiapoptotic gene Bcl-2 has been previously shown to protect cells from undergoing apoptosis during exposure to environmental stress. There is strong evidence that, in addition to its well-known effects on apoptosis, Bcl-2 is involved in antioxidant protection and regulation of cell cycle progression. To determine if the overexpression of Bcl-2 could improve the process of adaptation to suspension and protein-free growth environments, we have studied the growth and viability of anchorage-dependent Chinese hamster ovary cell lines that differ only in there expression of Bcl-2. In addition, we examined the effect of combining Bcl-2 and p21^CIP1 expression during adaptation to suspension and protein-free environments. The results of this study provide evidence of a clear reduction in the overall time required for the process of adaptation to both suspension and protein-free environments in Bcl-2 expressing cultures and that through the combined expression of p21^CIP1 and Bcl-2, it is possible to further reduce the time. The Bcl-2 results support the well-demonstrated concept that this protein plays an important role in apoptotic signaling pathways and suggest that it may also provide more diverse functions beside its death-inhibiting role.     

Integrating cell-cycle progression, drug penetration and energy metabolism to identify improved cancer therapeutic strategies

The effectiveness of chemotherapeutic drugs in tumors is reduced by multiple effects including drug diffusion and variable susceptibility of local cell populations. We hypothesized that quantifying the interactions between drugs and tumor microenvironments could be used to identify more effective anti-cancer strategies. To test this hypothesis we created a mathematical model that integrated intracellular metabolism, nutrient and drug diffusion, cell-cycle progression, cellular drug effects, and drug pharmacokinetics. To our knowledge, this is the first model that combines these elements and has coupled them to experimentally derived parameters. Drug cytotoxicity was assumed to be cell-cycle phase specific, and progression through the cell cycle was assumed to be dependent on ATP generation. The model consisted of a coupled set of nonlinear partial differential, ordinary differential and algebraic equations with an outer free boundary, which was solved using orthogonal collocation on a moving grid of finite elements. Model simulations showed the existence of an optimum drug diffusion coefficient: a low diffusivity prevents effective penetration before the drug is cleared from the blood and a high diffusivity limits drug retention. This result suggests that increasing the molecular weight of the anti-cancer drug paclitaxel from 854 to approximately 20,000 by nano-particle conjugation would improve its efficacy. The simulations also showed that fast growing tumors are less responsive to therapy than are slower tumors with more quiescent cells, demonstrating the competing effects of regrowth and cytotoxicity. The therapeutic implications of the simulation results are that (1) monolayer cultures are inadequate for accurately determining therapeutic effects in vitro, (2) decreasing the diffusivity of paclitaxel could increase its efficacy, and (3) measuring the proliferation fraction in tumors could enhance the prediction of therapeutic efficacy.     

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.     

miR-1290对人脐带间充质干细胞增殖、周期和凋亡的调控作用研究

目的:探索microRNA-1290对人脐带间充质干细胞增殖、细胞周期和凋亡的影响。方法:以从胎儿脐带分离的间充质干细胞为基础细胞,构建miR-1290模拟物,进行瞬时转染,通过荧光定量PCR检测转染后miR-1290水平;运用MTT方法观察转染miR-1290后间充质干细胞的增殖情况;通过流式细胞仪染色观察转染后细胞周期和细胞凋亡的变化。结果:荧光定量PCR结果显示,转染后miR-1290表达水平显著升高12.2倍;MTT结果表明,高表达miR-1290相比于阴性对照组(NC组)可以促进间充质干细胞的增殖;流式细胞周期检测结果发现,转染miR-1290后,细胞周期G1期从(85.90±1.91)%缩短至(76.35±2.03)%,而S期和G2期与对照组相比均明显增加,增殖指数(22.75±3.01)相比于对照组(14.10±1.87)也显著升高(P0.05);凋亡检测结果显示,高表达miR-1290细胞凋亡率(4.9±0.276)%与阴性对照组(8.2±0.891)%相比下降,有统计学意义(P0.05)。结论:miR-1290可以提高MSC的增殖能力,延长S期和G2期,并一定程度上抑制MSC的凋亡。     

Bub1基因对人肝癌MHCC97-H细胞增殖、周期和凋亡的影响

目的:研究Bub1基因在肝癌中的表达以及对肝癌细胞系MHCC97-H增殖、周期和凋亡的影响。方法:利用RNA干扰技术下调肝癌细胞系MHCC97-H中Bub1的表达;qRT-PCR和Western Blot分别检测Bub1在mRNA和蛋白水平表达的变化;CCK-8实验检测肿瘤细胞增殖能力的改变;流式细胞术检测细胞周期和凋亡的变化。结果:qRT-PCR和Western Blot结果显示si-Bub1能够成功下调Bub1的表达;下调Bub1后肝癌MHCC97-H细胞的增殖能力下降(P0.05),细胞的凋亡比例升高(P0.05),细胞发生S期阻滞。结论:Bub1基因在肝癌中高表达,下调Bub1的表达后能够降低肝癌细胞的增殖能力,促进细胞凋亡,诱导细胞发生S期阻滞。     

Combining Gompertzian growth and cell population dynamics

A two-compartment model of cancer cells population dynamics proposed by Gyllenberg and Webb includes transition rates between proliferating and quiescent cells as non-specified functions of the total population, N. We define the net inter-compartmental transition rate function: Phi(N). We assume that the total cell population follows the Gompertz growth model, as it is most often empirically found and derive Phi(N). The Gyllenberg-Webb transition functions are shown to be characteristically related through Phi(N). Effectively, this leads to a hybrid model for which we find the explicit analytical solutions for proliferating and quiescent cell populations, and the relations among model parameters. Several classes of solutions are examined. Our model predicts that the number of proliferating cells may increase along with the total number of cells, but the proliferating fraction appears to be a continuously decreasing function. The net transition rate of cells is shown to retain direction from the proliferating into the quiescent compartment. The death rate parameter for quiescent cell population is shown to be a factor in determining the proliferation level for a particular Gompertz growth curve.     

Polycystin-1 induced apoptosis and cell cycle arrest in G0/G1 phase in cancer cells

Studies have shown that polycystin-1, encoded by PKD1, the major ADPKD, may have a central role in regulating both apoptosis and proliferation, which could prevent the malignant transformation of affected cells. However, as a putative tumor suppressor, direct studies on the possibility that polycystin-1 may play a role in cancer cells' biological properties have not yet been reported. We have demonstrated that the apoptosis of cancer cells was induced by overexpression of polycystin-1. After transfection with polycystin-1, three cancer cell lines, HepG2, A549, and SW480, showed significantly increased apoptosis compared with the respective control groups. This was accompanied by cell cycle arrest at G(0)/G(1) phase, whereas cell proliferation was not significantly affected. Overexpression of polycystin-1 induces apoptosis in cancer cells, at least partially, through Wnt and a caspase-dependent pathway.     

Identification of a novel aFGF-binding peptide with anti-tumor effect on breast cancer from phage display library

It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.     

A pharmacologically based multiscale mathematical model of angiogenesis and its use in investigating the efficacy of a new cancer treatment strategy

Tumor angiogenesis is the process by which new blood vessels are formed and enhance the oxygenation and growth of tumors. As angiogenesis is recognized as being a critical event in cancer development, considerable efforts have been made to identify inhibitors of this process. Cytostatic treatments that target the molecular events of the angiogenesis process have been developed, and have met with some success. However, it is usually difficult to preclinically assess the effectiveness of targeted therapies, and apparently promising compounds sometimes fail in clinical trials.We have developed a multiscale mathematical model of angiogenesis and tumor growth. At the molecular level, the model focuses on molecular competition between pro- and anti-angiogenic substances modeled on the basis of pharmacological laws. At the tissue scale, the model uses partial differential equations to describe the spatio-temporal changes in cancer cells during three stages of the cell cycle, as well as those of the endothelial cells that constitute the blood vessel walls.This model is used to qualitatively assess how efficient endostatin gene therapy is. Endostatin is an anti-angiogenic endogenous substance. The gene therapy entails overexpressing endostatin in the tumor and in the surrounding tissue. Simulations show that there is a critical treatment dose below which increasing the duration of treatment leads to a loss of efficacy.This theoretical model may be useful to evaluate the efficacy of therapies targeting angiogenesis, and could therefore contribute to designing prospective clinical trials.     

Antiproliferative and apoptosis-inducing activity of schisandrin B against human glioma cells

Background

Malignant glioma is the most devastating and aggressive tumour in the brain and is characterised by high morbidity, high mortality and extremely poor prognosis. The main purpose of the present study was to investigate the effects of schisandrin B (Sch B) on glioma cells both in vitro and in vivo and to explore the possible anticancer mechanism underlying Sch B-induced apoptosis and cell cycle arrest.

Methods

The anti-proliferative ability of Sch B on glioma cells were assessed by MTT and clony formation assays. Flow cytometric analysis was used to detect cell cycle changes. Apoptosis was determined by Hoechst 33342 staining and annexin V/PI double-staining assays. The mitochondrial membrane potential was detected by Rhodamine 123 staining. The in vivo efficacy of Sch B was measured using a U87 xenograft model in nude mice. The expressions of the apoptosis-related and cell cycle-related proteins were analysed by western blot. Student’s t-test was used to compare differences between treated groups and their controls.

Results

We found that Sch B inhibited growth in a dose- and time-dependent manner as assessed by MTT assay. In U87 and U251 cells, the number of clones was strongly suppressed by Sch B. Flow cytometric analysis revealed that Sch B induced cell cycle arrest in glioma cells at the G0/G1 phase. In addition, Sch B induced glioma cell apoptosis and reduced mitochondrial membrane potential (ΔΨm) in a dose-dependent manner. Mechanically, western blot analysis indicated that Sch B induced apoptosis by caspase-3, caspase-9, PARP, and Bcl-2 activation. Moreover, Sch B significantly inhibited tumour growth in vivo following the subcutaneous inoculation of U87 cells in athymic nude mice.

Coclusions

In summary, Sch B can reduce cell proliferation and induce apoptosis in glioma cells and has potential as a novel anti-tumour therapy to treat gliomas.     

Estrogen receptor beta in the breast: role in estrogen responsiveness and development of breast cancer

Breast cancer is one of the most common forms of cancer observed in women. Endogenous estrogen is thought to play a major role in its development and estrogen receptor blockers are the most important drugs in its treatment. It has long been thought that any conditions or exposures, which enhance estrogenic responses, would result in an increased risk for breast cancer. The discovery of the second estrogen receptor, ERbeta, which can have effects opposite to those of the well-known 'original' estrogen receptor (now called ERalpha) challenges this simplistic view. In order to understand breast cancer one must first understand how the normal breast is maintained. The functions of ERbeta in the breast remain to be defined but from what we have learnt about its activities in in vitro systems, this estrogen receptor may have a protective role in the breast. Studies in human and rodent breasts as well as in human breast cancer biopsies reveal that ERbeta is by far the more abundant of the two ERs. Despite the role of estrogen in proliferation of the breast, neither of the two ERs appears to located in epithelial cells which divide in response to estrogen. In order to define the functions of ERbeta in the normal and malignant breast, we have created mice in which the ERbeta gene has been inactivated. Studies of the breasts of ERbeta knock out mice (BERKO) revealed abnormal epithelial growth, overexpression of Ki67 and severe cystic breast disease as mice age.     

Synthesis and characterization of new copper thiosemicarbazone complexes with an ONNS quadridentate system: cell growth inhibition, S-phase cell cycle arrest and proapoptotic activities on cisplatin-resistant neuroblastoma cells

Two new copper thiosemicarbazone complexes with an ONNS quadridentate system were synthesized and evaluated for anticancer activity on cisplatin-resistant neuroblastoma cells. Among these two copper complexes, the substituted 8-hydroxyquinoline-2-carboxaldehyde–4,4-dimethyl-3-thiosemicarbazide (CuHQDMTS) exhibited stronger cell growth inhibition activity than the unsubstituted copper 8-hydroxyquinoline-2-carboxaldehyde thiosemicarbazide complex (CuHQTS). Both CuHQTS and CuHQDMTS showed dose-dependent cell growth inhibition, cell cycle arrest and apoptosis induction activities on the SK-N-DZ neuroblastoma cells. Increased expression of p53 protein molecules was detected in the SK-N-DZ cells treated with CuHQTS. The data obtained in this study suggest that CuHQDMTS and CuHQTS hold potential as new, effective drugs for treatment of refractory neuroblastoma in children.     

ATAD3A在结直肠癌组织中的表达及其对细胞生长的影响

目的:探讨三磷酸腺苷酶家族蛋白3A(ATAD3A)在结直肠癌组织中的表达情况,并验证其对结直肠癌细胞RKO和HCT116生长的影响。方法:收集结直肠癌患者配对癌与癌旁组织115例,通过免疫组化方式验证ATAD3A在结直肠癌组织与癌旁的表达差异。采用慢病毒转染和si-RNA干涉的方式构建ATAD3A过表达和敲低肠癌细胞系,并采用MTS,流式检测细胞周期和细胞凋亡等方法验证ATAD3A对结直肠癌细胞系RKO和HCT116的影响。结果:ATAD3A在结直肠癌组织中表达较癌旁组织显著升高(P0.001)。在结直肠癌细胞系RKO和HCT116中过表达ATAD3A后,细胞增殖能力明显增强,处于S期的细胞比例明显增加,而且细胞凋亡数量明显减少。反之,在上述肠癌细胞中干涉ATAD3A后,细胞增殖能力减弱,细胞大部分停滞于G1期,而且凋亡细胞数量明显增多。结论:ATAD3A在结直肠癌组织中表达升高,且ATAD3A通过促进细胞增殖、细胞周期进程和抑制细胞凋亡等方式促进肠癌细胞的生长。     

Proscillaridin A induces apoptosis and inhibits the metastasis of osteosarcoma in vitro and in vivo

The side effects of chemotherapy, drug resistance, and tumor metastasis hinder the development of treatment for osteosarcoma, leading to poor prognosis of patients with the disease. Proscillaridin A, a kind of cardiac glycoside, has been proven to have anti-proliferative properties in many malignant tumors, but the efficacy of the drug in treating osteosarcoma is unclear. In the present study, we assessed the effects of Proscillaridin A on osteosarcoma and investigated its underlying action mechanism. The cell cytotoxicity assay showed that Proscillaridin A significantly inhibited the proliferation of 143B cells in a dose- and time-dependent manner. Also, flow cytometry and invasion assay revealed that Proscillaridin A induced apoptosis and reduced 143B cell motility. Western blotting and PCR were used to detect the expressions of Bcl-xl and MMP2 and showed that mRNA/protein expression levels decreased significantly in Proscillaridin A-treated osteosarcoma cells. Using a mouse xenograft model, we found that Proscillaridin A treatment significantly inhibited tumor growth and lung metastasis in vivo and decreased the expression levels of Bcl-xl and MMP2. No noticeable side effect was observed in the liver, kidney, and hematological functions. Conclusively, Proscillaridin A suppressed proliferation, induced apoptosis, and inhibited 143B cell metastasis in vitro and in vivo, and these effects could be mediated by downregulating the expressions of Bcl-xl and MMP2.     

17beta-estradiol affects proliferation and apoptosis of rat prostatic smooth muscle cells by modulating cell cycle transition and related proteins

Abundant evidence indicates that estrogens have an important role in the pathology of benign prostatic hyperplasia (BPH). To investigate the effect of 17beta-estradiol (E2) on the proliferation and apoptosis of prostatic smooth muscle cells (PSMCs), rat PSMCs were obtained and exposed to gradient concentrations (0.1-100 nmol/l) of E2 over varying amounts of time. The progression of cell cycle, cellular apoptosis, cyclin D1, Bcl-2 and Bax proteins were detected. The data show that the effect of E2 on rat PSMCs is bilateral: it promotes cell proliferation by enhancing the expression of cyclin D1, which accelerates G1 to S phase transition; on the other hand, it induces apoptosis of the cells by up-regulating the expression of Bax. We thus suggest that an increase in estrogen may exert a launching effect in the pathology of BPH.     

Inhibition of proliferation and induction of apoptosis by abrogation of heat-shock protein (HSP) 70 expression in tumor cells

Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to invesitgate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidiumiodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.