2-Deoxyglucose combined with wild-type p53 overexpression enhances cytotoxicity in human prostate cancer cells via oxidative stress

Overexpression of the tumor suppressor gene, wild-type p53 (wtp53), using adenoviral vectors (Adp53) has been suggested to kill cancer cells by hydroperoxide-mediated oxidative stress [1,2] and nutrient distress induced by the glucose analog, 2-deoxyglucose (2DG), has been suggested to enhance tumor cell killing by agents that induce oxidative stress via disrupting hydroperoxide metabolism [3,4]. In the current study clonogenic cell killing of PC-3 and DU-145 human prostate cancer cells (lacking functional p53) mediated by 4 h exposure to 50 plaque forming units (pfus)/cell of Adp53 (that caused the enforced overexpression of wtp53) was significantly enhanced by treatment with 2DG. Accumulation of glutathione disulfide was found to be significantly greater in both cell lines treated with 2DG+Adp53 and both cell lines treated with 2DG+Adp53 showed a approximately 2-fold increases in dihydroethidine (DHE) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CDCFH(2)) oxidation, indicative of increased steady-state levels of O(2)(.-) and hydroperoxides, respectively. Finally, overexpression of catalase or glutathione peroxidase using adenoviral vectors partially, but significantly, protected DU-145 cells from the toxicity induced by 2DG+Adp53 treatment. These results show that treatment of human prostate cancer cells with the combination of 2DG (a nutrient stress) and overexpression of the tumor suppressor gene, wtp53, enhances clonogenic cell killing by a mechanism that involves oxidative stress as well as allowing for the speculation that inhibitors of glucose and hydroperoxide metabolism can be used in combination with Adp53 gene therapy to enhance therapeutic responses.     

PTEN protects p53 from Mdm2 and sensitizes cancer cells to chemotherapy.

The PTEN tumor suppressor protein inhibits phosphatidylinositol 3-kinase (PI3K)/Akt signaling that promotes translocation of Mdm2 into the nucleus. When restricted to the cytoplasm, Mdm2 is degraded. The ability of PTEN to inhibit the nuclear entry of Mdm2 increases the cellular content and transactivation of the p53 tumor suppressor protein. Retroviral transduction of PTEN into U87MG (PTEN null) glioblastoma cells increases p53 activity and expression of p53 target genes and induces cell cycle arrest. U87MG/PTEN glioblastoma cells are more sensitive than U87MG/PTEN null cells to death induced by etoposide, a chemotherapeutic agent that induces DNA damage. Previously, tumor suppressor proteins have been supposed to act individually to suppress cancers. Our results establish a direct connection between the activities of two major tumor suppressors and show that they act together to respond to stresses and malignancies. PTEN protects p53 from survival signals, permitting p53 to function as a guardian of the genome. By virtue of its capacity to protect p53, PTEN can sensitize tumor cells to chemotherapy that relies on p53 activity. p53 induces PTEN gene expression, and here it is shown that PTEN protects p53, indicating that a positive feedback loop may amplify the cellular response to stress, damage, and cancer.     

Transduction of Apaf-1 or caspase-9 induces apoptosis in A-172 cells that are resistant to p53-mediated apoptosis

p53 replacement gene therapy has been carried out clinically for cancers with p53 mutations; however, some cancers are resistant to p53 gene therapy. In this study, we transduced A-172 and U251 cells harboring p53 mutations with wild-type p53 using adenovirus vectors to induce wild-type p53 protein at similar expression levels. A-172 cells did not undergo apoptosis after p53 transduction, whereas U251 cells were markedly sensitive to p53-mediated apoptosis. A-172 cells showed higher endogenous expression of Bcl-X(L) than U251, and transduction of Bcl-X(L) repressed p53-mediated apoptosis in U251 cells, suggesting that high endogenous expression of Bcl-X(L) renders A-172 cells, at least in part, resistant to p53-mediated apoptosis. We transduced A-172 cells and U251 cells with the Apaf-1 or caspase-9 genes; both are downstream components of p53-mediated apoptosis. We found that A-172 cells were highly sensitive to Apaf-1- and caspase-9-mediated apoptosis. The results indicate that A-172 cells harboring mutant p53 were not susceptible to p53-mediated apoptosis, possibly due to high endogenous expression of Bcl-X(L). Transduction of Apaf-1 or caspase-9 would override the resistance mechanism of apoptosis in A-172 cells. These findings provide potentially a novel approach in killing cancers that are resistant to p53 replacement gene therapy.     

Induction of p53 protein expression by sodium arsenite

Arsenic is carcinogen for humans and has been shown to act as an enhancer in initiated animal models. In a previous work we found impairment of lymphocyte proliferation in arsenic-exposed individuals and in vitro we obtained dose-related inhibition of mitotic response and lymphocyte proliferation. Intrigued by these effects and based on the role of p53 on cell proliferation, we tested different concentrations of sodium arsenite for their ability to induce the expression of tumor suppressor gene p53 in different cell lines (HeLa, C-33A, Jurkat) and a lymphoblast cell line transformed with Epstein–Barr virus (LCL-EBV). We also evaluated changes in their viability after 24 h arsenic treatment; C-33A cells showed the higher sensitivity to arsenic treatment while HeLa, Jurkat and LCL-EBV cells showed similar cytotoxicity curves. Immunoblots showed an increased expression of p53 gene with 1 μM sodium arsenite in Jurkat cells and 10 μM sodium arsenite in HeLa and LCL-EBV cells. In addition, we transfected Jurkat cells and human lymphocytes with wild-type and mutated p53 genes; lymphocytes and Jurkat cells that received the mutated p53 showed increased sensitivity to arsenic cytotoxicity. Data obtained indicate that arsenic induces p53 expression and that cells with a functional p53 contend better with damage induced by this metalloid.     

Expression of p16(INK4A) induces dominant suppression of glioblastoma growth in situ through necrosis and cell cycle arrest

Tumor suppressor genes may represent an important new therapeutic modality in the treatment of human glioblastoma (GBM). p16(INK4A) is a tumor suppressor gene with mutation and/or deletion found in many human tumors, including glioblastomas, melanoma, and leukemias. RT-2 rat GBM cell line was used to investigate if the p16 gene induces dominant suppression of glioblastoma growth. Close to 100% of tumor cells were infected by high titer pCL retrovirus encoding the full-length human p16 cDNA at 5 m.o.i. Infected cells showed a 98% reduction in colony forming assay and a 60% reduction in growth curves in vitro compared to vector control. Exogenous overexpression of p16 induced hypophosphorylation of Rb protein by Western blot analysis. Intracranial injection of p16-infected tumor cells into syngeneic rats resulted in a 95% reduction in tumor volume compared to the controls. Intratumoral injection of p16 retrovirus resulted in tumor necrosis and prominent human p16 transgene expressions. Proliferation marker PCNA was not detected in these human p16-expressed RT-2 tumor cells, suggesting the cells were unable to enter into S phase after p16 expression. In addition, direct repeat intracranial injections of p16 retrovirus prolonged animal survival 3.2-fold compared to the controls (48.4 +/- 13.4 vs 15.0 +/- 2.1 days, p < 0.001). Two out of ten rats were found with dormant tumors at day 60 after p16 retrovirus injection. These results showed that p16 is effective in inhibiting GBM growth in situ. The mechanisms of tumor growth reduction and necrosis in vivo might be due to G1 arrest triggered by p16 expression.     

Comparisons of tumor suppressor p53, p21, and p16 gene therapy effects on glioblastoma tumorigenicity in situ

The mutation and/or deletion of tumor suppressor genes have been postulated to play a major role in the genesis and the progression of gliomas. In this study, the functional expression and efficacy in tumor suppression of 3 tumor suppressor genes (p53, p21, and p16) were tested and compared in a rat GBM cell line (RT-2) after retrovirus mediated gene delivery in vitro and in vivo. Significant reductions in tumor cell growth rate were found in p16 and p21 infected cells (60 +/- 12% vs 66 +/- 15%) compared to p53 (35 +/- 9%). In vitro colony formation assay also showed significant reductions after p16 and p21 gene delivery (98 +/- 5% vs 91 +/- 10%) compared to p53 (50 +/- 18%). In addition, the tumor suppression efficacy were investigated and compared in vivo. Retroviral mediated p16 and p21 gene deliveries in glioblastomas resulted in more than 90% reductions in tumor growth (92 +/- 26% vs 90 +/- 22%) compared to p53 (62 +/- 18%). Tumor suppressor gene insertions in situ further prolonged animal survival. Overall p16 and p21 genes showed more powerful tumor suppressor effects than p53. The results were not surprising, as p16 and p21 are more downstream in the cell cycle regulatory pathway compared to p53. Moreover, the mechanism involved in each of their suppressor effects is different. This study demonstrates the feasibility of using tumor suppressor genes in regulating the growth of glioma in vitro and in situ.     

Tet 调控的脑相对特异性新基因 LRRC4表达细胞系的构建

LRRC4是一个在脑相对特异性表达的富亮氨酸重复超家族新成员,在神经胶质瘤表达明显下调或缺失且具有抑制脑胶质瘤细胞生长的潜能. 利用 Tet-on 基因表达系统,经过两轮转染,先后将调控质粒 pTet-on 和表达质粒 pTRE-2hyg-LRRC4 转染 U251 细胞系,分别用 G418 和潮霉素 Hygromycin 进行两次筛选. 在第一轮挑取的 80 个克隆中,利用 pTRE-2hyg-luciferase 报告基因进行最佳的低背景高表达的 pTet-on 细胞克隆筛选,在通过量效关系和动力学检测筛选的最佳克隆基础上,再进行 pTRE-2hyg-LRRC4 的转染,并通过 RT-PCR 和 RNA 印迹检测,成功获得了两个具有良好诱导性 Tet 调控的 LRRC4 双稳定表达细胞系,为进一步阐明 LRRC4 在脑胶质瘤发生发展中的作用,提供有利的研究基础和理想的实验平台.     

A novel adenoviral vector expressing human Fas/CD95/APO-1 enhances p53-mediated apoptosis.

Recent evidence suggests an intriguing link between p53 and the Fas pathway. To evaluate this association further, we utilized a recombinant adenoviral vector (AdWTp53) to overexpress wild-type p53 in lung cancer (A549, H23, EKVX and HOP92) and breast cancer (MDA-MB-231 and MCF-7) cell lines and observed an increase in the Fas/CD95/APO-1 protein levels. Furthermore, this increase correlated with the sensitivity of the cell lines to p53-mediated cytotoxicity. To examine the effects of Fas over-expression in cells resistant to p53 over-expression, we constructed AdFas, an adenoviral vector capable of transferring functional human Fas to cancer cells. Interestingly, infection of p53-resistant MCF-7 cells with AdFas sensitized them to p53-mediated apoptosis. These studies indicate that combined over-expression of Fas and wild-type p53 may be an effective cancer gene therapy approach, especially in cells relatively resistant to p53 over-expression.     

Taiwanin A induced cell cycle arrest and p53-dependent apoptosis in human hepatocellular carcinoma HepG2 cells

Taiwanin A, a lignan isolated from Taiwania cryptomerioides Hayata, has previously been reported to have cytotoxicity against human tumor cells, but the mechanisms are unclear. In this study, we examined the molecular mechanism of cell death of human hepatocellular carcinoma HepG2 cells induced by Taiwanin A. Taiwanin A has been found to induce cell cycle arrest at G2/M phase as well as caspase-3-dependent apoptosis within 24 h. We performed both in vitro turbidity assay and immunofluorescence staining of tubulin to show that Taiwanin A can inhibit microtubule assembly. Moreover, the tumor suppressor protein p53 in HepG2 cells was activated by Taiwanin A within 12 h. Inhibition of p53 by either pifithrin-alpha or by short hairpin RNA which blocks p53 expression attenuates Taiwanin A cytotoxicity. Our results demonstrate that Taiwanin A can act as a new class of microtubule damaging agent, arresting cell cycle progression at mitotic phase and inducing apoptosis through the activation of p53.     

Polo-like kinase 1 inhibitors,mitotic stress and the tumor suppressor p53

Polo-like kinase 1 has been established as one of the most attractive targets for molecular cancer therapy. In fact, multiple small-molecule inhibitors targeting this kinase have been developed and intensively investigated. Recently, it has been reported that the cytotoxicity induced by Plk1 inhibition is elevated in cancer cells with inactive p53, leading to the hypothesis that inactive p53 is a predictive marker for the response of Plk1 inhibition. In our previous study based on different cancer cell lines, we showed that cancer cells with wild type p53 were more sensitive to Plk1 inhibition by inducing more apoptosis, compared with cancer cells depleted of p53. In the present work, we further demonstrate that in the presence of mitotic stress induced by different agents, Plk1 inhibitors strongly induced apoptosis in HCT116 p53^+/+ cells, whereas HCT116 p53^−/− cells arrested in mitosis with less apoptosis. Depletion of p53 in HCT116 p53^+/+ or U2OS cells reduced the induction of apoptosis. Moreover, the surviving HCT116 p53^−/− cells showed DNA damage and a strong capability of colony formation. Plk1 inhibition in combination with other anti-mitotic agents inhibited proliferation of tumor cells more strongly than Plk1 inhibition alone. Taken together, the data underscore that functional p53 strengthens the efficacy of Plk1 inhibition alone or in combination by strongly activating cell death signaling pathways. Further studies are required to investigate if the long-term outcomes of losing p53, such as low differential grade of tumor cells or defective DNA damage checkpoint, are responsible for the cytotoxicity of Plk1 inhibition.     

Tumor antigen LRRC15 impedes adenoviral infection: implications for virus-based cancer therapy

Adenoviruses for gene or oncolytic therapy are under development. Notable among these strategies is adenoviral delivery of the tumor suppressor p53. Since all therapeutics have limitations in certain settings, we have undertaken retroviral suppressor screens to identify genes conferring resistance to adenovirus-delivered p53. These studies identified the tumor antigen LRRC15, which is frequently overexpressed in multiple tumor types, as a repressor of cell death due to adenoviral p53. LRRC15, however, does not impede p53 function per se but impedes adenoviral infection. Specifically, LRRC15 causes redistribution of the coxsackievirus-adenovirus receptor away from the cell surface. This effect is manifested in less adenoviral binding to the surfaces of LRRC15-expressing cells. This discovery, therefore, not only is important for understanding adenoviral biology but also has potentially important implications for adenovirus-based anticancer therapeutics.     

Effect of G-rich oligonucleotides on the proliferation of leukemia cells and its relationship with p53 expression

G-rich oligonucleotides (GROs) can inhibit cell proliferation by inducing cell cycle arrest at S phase in tumor cell lines. GROs bind specific cellular proteins, such as nucleolin, a crucial protein interacting with P53; however, little is known about the relationship between GROs and P53. In this study, we have shown that GROs inhibited the proliferation of U937 cells (a human monocytic leukemia cell line without P53 expression) by inducing S-phase arrest. We also showed that GRO colocalized with nucleolin in U937 cells. GRO treatment induced alteration of a series of cell cycle regulatory proteins in U937 cells. Increased Cdk2 expression might promote the cells to enter S phase and subsequent decrease of Cdk2 might induce cell cycle arrest in S phase. Transfection of U937 cells with a wild-type p53 gene caused the formation of nucleolin-P53 complex, which alleviated the effect of GRO on leukemia cells. This alleviated effect is probably due to the decreased uptake of GRO.     

EphA2对神经胶质瘤细胞系U251的凋亡﹑增殖﹑迁移和侵袭的研究

为了研究EphA2对神经胶质瘤细胞系U251在增殖、凋亡、迁移和侵袭方面所起的作用,用RT-PCR方法检测正常脑组织标本与两种恶性胶质瘤细胞系中EphA2 mRNA表达水平,然后用化学合成的针对EphA2基因的小干扰RNA(siRNA)下调该基因的表达,以检测其在U251中的生物学功能．证实了EphA2基因在正常脑组织标本中的表达水平远低于两种恶性胶质瘤细胞系．把体外化学合成针对EphA2基因的小干扰RNA(siRNA- EphA2)转染入U251细胞后,Western blot, 实时定量 RT-PCR检测到U251细胞中EphA2蛋白及mRNA表达水平都明显降低,并且细胞增殖受到显著抑制,同时出现了明显的细胞凋亡．伤口愈合实验(检测细胞迁移能力),Transwell小室实验(检测细胞侵袭能力)均表明,下调EphA2的表达后,细胞的迁移和侵袭能力较阴性对照组显著减弱．上述结果表明,在神经胶质瘤U251细胞中,EphA2与其恶性增殖及高度侵染性相关,可作为分子治疗的有效靶点．     

RNA interference-mediated silencing of iASPP induces cell proliferation inhibition and G0/G1 cell cycle arrest in U251 human glioblastoma cells

iASPP is an evolutionally conserved inhibitory member of the ASPP (apoptosis-stimulating protein of p53) protein family. Overexpression of iASPP was observed in several types of human tumors, however, its role in tumorigenesis has not been fully clarified. To investigate the role of iASPP in human glioblastoma multiforme (GMB) progression, the authors employed lentivirus-mediated shRNA to silence endogenous iASPP expression and elucidated iASPP function by analysis of viability, colony formation, DNA synthesis, and cell cycle in p53-mutant glioblastoma cell line U251. iASPP was significantly and sustainably knocked down by iASPP-specific shRNA in U251 cells. Stable down-regulation of iASPP expression-induced cell proliferation inhibition and G0/G1 cell cycle arrest by down-regulation of cyclin D1 and up-regulation of p21(Waf1/Cip1). Thus, the findings not only provide a molecular basis for the role of iASPP in cell cycle progression of glioblastoma cells but also suggest a novel therapeutic target for the treatment of GBM.     

Induction of G1 Cell Cycle Arrest in Human Glioma Cells by Salinomycin Through Triggering ROS-Mediated DNA Damage In Vitro and In Vivo

Chemotherapy has always been one of the most effective ways in combating human glioma. However, the high metastatic potential and resistance toward standard chemotherapy severely hindered the chemotherapy outcomes. Hence, searching effective chemotherapy drugs and clarifying its mechanism are of great significance. Salinomycin an antibiotic shows novel anticancer potential against several human tumors, including human glioma, but its mechanism against human glioma cells has not been fully elucidated. In the present study, we demonstrated that salinomycin treatment time- and dose-dependently inhibited U251 and U87 cells growth. Mechanically, salinomycin-induced cell growth inhibition against human glioma was mainly achieved by induction of G1-phase arrest via triggering reactive oxide species (ROS)-mediated DNA damage, as convinced by the activation of histone, p53, p21 and p27. Furthermore, inhibition of ROS accumulation effectively attenuated salinomycin-induced DNA damage and G1 cell cycle arrest, and eventually reversed salinomycin-induced cytotoxicity. Importantly, salinomycin treatment also significantly inhibited the U251 tumor xenograft growth in vivo through triggering DNA damage-mediated cell cycle arrest with involvement of inhibiting cell proliferation and angiogenesis. The results above validated the potential of salinomycin-based chemotherapy against human glioma.