Ginsenoside Rh2 induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53

Ginsenosides are the main bioactive components in American ginseng, a commonly used herb. In this study, we showed that the ginsenoside Rh2 exhibited significantly more potent cell death activity than the ginsenoside Rg3 in HCT116 and SW480 colorectal cancer cells. Cell death induced by Rh2 is mediated in part by the caspase-dependent apoptosis and in part by the caspase-independent paraptosis, a type of cell death that is characterized by the accumulation of cytoplasmic vacuoles. Treatment of cells with Rh2 activated the p53 pathway and significantly increased the levels of the pro-apoptotic regulator, Bax, while decreasing the levels of anti-apoptosis regulator Bcl-2. Removal of p53 significantly blocked Rh2-induced cell death as well as vacuole formation, suggesting that both types of cell death induced by Rh2 are mediated by p53 activity. Furthermore, we show that Rh2 increased ROS levels and activated the NF-κB survival pathway. Blockage of ROS by NAC or catalase inhibited the activation of NF-κB signaling and enhanced Rh2-induced cell death, suggesting that the anti-cancer effect of Rh2 can be enhanced by antioxidants.     

Fas-mediated apoptosis is dependent on wild-type p53 status in human cancer cells expressing a temperature-sensitive p53 mutant alanine-143

The p53 mutant 143Ala is a human temperature-sensitive mutant with two conformational states.To definitively determine whether the Fas signal transduction pathway and the function of the pathway are dependent on p53 status, we have established stable transfectants of p53 mutant 143Ala in two human cancer cell lines: H1299 (lung cancer line) and PC-3 (prostate cancer line), the native state of which contains null p53 status and can grow at 37 degrees C and 32.5 degrees C. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle analysis showed inhibition of the growth of cells overexpressing p53 mutant 143Ala in the wild-type p53 form at 32.5 degrees C because of induction of G0/G1 arrest. Transfected cells had increased protein expression of p21, Fas, and MDM2 at the wild-type p53 conformation at 32.5 degrees C, but not in the mutant p53 form at 37 degrees C. However, there was no change in protein expression of FADD, FAP-1, Bcl-2, or Bax at 32.5 or 37 degrees C. Assays for apoptosis demonstrated that anti-Fas antibody CH-11 and FasL induced apoptosis only in cells that overexpress p53 mutant 143Ala at 32.5 degrees C with the wild-type p53 form. Both caspase-3 and caspase-8 activities were increased by anti-Fas antibody CH-11 only in cells at 32.5 degrees C with wild-type p53. Our results demonstrated that Fas-mediated apoptosis in H1299 and PC-3 cells expressing p53 mutant 143Ala occurred only with the wild-type p53 phenotype. These results support the hypothesis that Fas-mediated apoptosis is dependent, at least partially, on the presence of a functional wild-type p53 state. This model may be a useful tool for dissecting the specific interactions between wild-type p53 and the Fas signal transduction pathway in human cancer cells.     

Myc and E2F1 induce p53 through p14ARF-independent mechanisms in human fibroblasts

p19ARF is induced in response to oncogene activation or during cellular senescence in mouse embryo fibroblasts, triggering p53-dependent and p53-independent cell cycle arrest and apoptosis. We have studied the involvement of human p14ARF as a regulator of p53 activity in normal human skin fibroblasts (NHFs) or WI38 lung embryonic fibroblasts expressing conditional Myc or E2F1 estrogen receptor fusion proteins. Both Myc and E2F1 activation rapidly induced p53 phosphorylation at Ser-15, p53 protein accumulation, and upregulation of the p53 target genes MDM2 and p21. Activation of E2F1 induced p14ARF mRNA and protein levels. In contrast, Myc activation did not induce any significant increase in p14ARF mRNA or protein levels in neither NHFs nor WI38 fibroblasts within 48 h. Myc and E2F1 induced p53 and cell cycle arrest even after silencing of p14ARF using short-interfering RNA. Treatment with the ATM/ATR kinase inhibitor caffeine prevented p53 accumulation upon activation of Myc or E2F1. Our results indicate that p53 phosphorylation, but not p14ARF, plays a major role for the induction of p53 in response to Myc and E2F1 activation in normal human fibroblasts.     

Therapeutic potential of recombinant p53 overexpression in breast cancer cells expressing endogenous wild-type p53

Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21^{WAF 1/CIP1} protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.     

低氧条件下食管癌细胞中PRRX1通过p53介导线粒体通路诱导细胞凋亡的研究

目的:探究低氧条件下配对相关同源框1（PRRX1）通过p53介导的线粒体通路诱导食管癌细胞凋亡。方法:GEPIA2数据库分析PRRX1基因在食管癌组织和正常食管组织中的表达变化。RT-qPCR和Western blotting分别检测HEEC、Eca-109、TE-1细胞中PRRX1的mRNA和蛋白表达。二氧化钴处理模拟低氧微环境,在常氧和低氧条件下检测Eca-109、TE-1细胞中PRRX1的mRNA和蛋白表达情况。采用小干扰RNA（Si-PRRX1）转染TE-1细胞,设置分组为Hypoxia-Si-NC、Hypoxia-Si-PRRX1。流式细胞术检测TE-1细胞凋亡,RT-qPCR检测p53 mRNA表达,Western blotting检测p53、BCL-2、BAX、Cleaved-Caspase3等蛋白表达,在TE-1-PRRX1 KO细胞系中过表达p53,检测 BCL-2、BAX、Cleaved-Caspase3等蛋白表达。结果:PRRX1在食管癌组织及细胞系中均高表达。在TE-1细胞中敲低PRRX1,抑制细胞凋亡,下调p53的mRNA及蛋白表达,抑制BAX、Cleaved-Caspase3蛋白表达,促进BCL-2蛋白表达;过表达PRRX1则上调p53蛋白表达。在TE-1-PRRX1 KO细胞系中过表达p53显著抑制BCL-2蛋白表达,促进BAX、Cleaved-Caspase3蛋白表达。结论:低氧条件下,PRRX1通过p53介导的线粒体通路诱导食管癌细胞凋亡。     

Arsenic trioxide induces apoptosis in human gastric cancer cells through up-regulation of p53 and activation of caspase-3

Arsenic trioxide (As(2)O(3)) can induce clinical remission in patients suffering from acute promyelocytic leukemia, through induction of apoptosis and activation of caspases. We investigated the potential use of As(2)O(3) in human gastric cancer and its possible mechanisms. Human gastric cancer cell lines AGS and MKN-28 were treated with various concentrations (0.1 to 100 microM) of As(2)O(3) for 24 to 72 hr. Apoptosis was determined by acridine orange staining, flow cytometry and DNA fragmentation. Protein levels of p53, p21(waf1/cip1), c-myc, bcl-2 and bax were detected by Western blotting. Effects of As(2)O(3) on caspase-3 protease activity, its protein concentration and cleavage of poly(ADP)-ribose polymerase (PARP) were also studied. As(2)O(3) inhibited cell growth and induced apoptosis in both cell lines, though AGS cells were more sensitive. As(2)O(3) induced apoptosis in AGS cells in a concentration- and time-dependent manner. Treatment resulted in a marked increase in p53 protein levels as early as 4 hr. Co-incubation with p53 anti-sense oligo-nucleotide suppressed As(2)O(3)-induced intracellular p53 over-expression and apoptosis. As(2)O(3) increased the activity of caspase-3, with appearance of its 17 kDa peptide fragment, and cleavage of PARP, with appearance of the 85 kDa cleavage product, both in parallel with the induction of apoptosis. Both the tripeptide caspase inhibitor zVAD-fmk and the specific caspase-3 inhibitor DEVD-fmk partially suppressed As(2)O(3)-induced caspase-3 activation and apoptosis. As(2)O(3) inhibits cell growth and induces apoptosis in gastric cancer cells, involving p53 over-expression and activation of caspase-3. The potential use of this compound in the treatment of gastric cancer is worth further investigation.     

siRNA-mediated down-regulation of iASPP promotes apoptosis induced by etoposide and daunorubicin in leukemia cells expressing wild-type p53

Oncoprotein inhibitory member of the ASPP family (iASPP) is a key inhibitor of tumor suppressor p53. Our previous study revealed that the expression of iASPP in acute leukemia (AL) patients was higher than that of normal control which implied that iASPP might play an important role in the pathogenesis and/or disease progression of AL. In this study, the iASPP expression was blocked by RNA interference (RNAi) in two leukemic cell lines, Nalm6 and K562, to explore the effects of iASPP on leukemia cells. The results indicated that down-regulation of endogenous iASPP increased p53-dependent apoptosis of leukemia cells. Thus, iASPP could be a molecular target in leukemia therapy.     

2-Methoxyestradiol induces p53-associated apoptosis of colorectal cancer cells

Menopausal estrogen replacement therapy is thought to be responsible for the recent decline in colorectal cancer (CRC) incidence among women. In the C57BL/6J-Min/+ mouse, an animal model of CRC, 17beta-estradiol (E(2)) prevents tumor formation in ovariectomized females. We examined human CRC intestinal cell lines to determine whether particular E(2) metabolites produced anti-tumor effects. Treatment of CRC cells with 2-methoxyestradiol (2-MeOE(2)) increased expression of p53 and p21(WAF1/CIP1) proteins and induced apoptosis, but did not produce changes in expression of estrogen receptor (ER)alpha or ERbeta. The finding that 2-MeOE(2) induces p53-mediated colon cell apoptosis in vitro supports a role for 2-MeOE(2) as an endogenous mediator of intestinal tumor suppression.     

Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells

Thymoquinone (TQ), the major biologically active component isolated from a traditional medicinal herb, Nigella sativa Linn, is a potential chemopreventive and chemotherapeutic compound. Despite the promising antineoplastic activities of TQ, the molecular mechanism of its pharmacologic effects is poorly understood. Here, we report that TQ exhibits antiproliferative effect, induces apoptosis, disrupts mitochondrial membrane potential and triggers the activation of caspases 8, 9 and 3 in myeloblastic leukemia HL-60 cells. The apoptosis induced by TQ was inhibited by a general caspase inhibitor, z-VAD-FMK; a caspase-3-specific inhibitor, z-DEVD-FMK; as well as a caspase-8-specific inhibitor, z-IETD-FMK. Moreover, the caspase-8 inhibitor blocked the TQ-induced activation of caspase-3, PARP cleavage and the release of cytochrome c from mitochondria into the cytoplasm. In addition, TQ treatment of HL-60 cells caused a marked increase in Bax/Bcl2 ratios due to upregulation of Bax and downregulation of Bcl2 proteins. These results indicate that TQ-induced apoptosis is associated with the activation of caspases 8, 9 and 3, with caspase-8 acting as an upstream activator. Activated caspase-8 initiates the release of cytochrome c during TQ-induced apoptosis. Overall, these results offer a potential mechanism for TQ-induced apoptosis in p53-null HL-60 cancer cells.     

Eicosapentaenoic acid induces Fas-mediated apoptosis through a p53-dependent pathway in hepatoma cells

Eicosapentaenoic acid (EPA) has been demonstrated to induce apoptosis and cell cycle arrest in various cancer cell lines in vitro. In this study, we investigated the anti-tumor effects of EPA on hepatoma cell lines and the mechanisms responsible for induced cell death. Three hepatoma cell lines tested had different p53 status: HepG2 with a wild-type p53; Hep3B, of which the endogenous p53 was deleted; and Huh7 with its p53 mutated. MTT assay showed reduced viability of HepG2 cells after exposure to EPA, and the cytotoxicity of EPA was time and dose dependent. However, EPA had no effect on the viability and cell death in the two other hepatoma cell lines containing dysfunctional p53. DNA fragmentation analysis and TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine diphosphate [dUTP] nick end labeling) staining showed a typical pattern of DNA laddering and DNA breaks staining, respectively, in wild-type p53-containing HepG2 cells after EPA treatment. We also observed that EPA induced transient nuclear accumulation of P53 protein that subsequently up-regulated the expression of Fas messenger RNA and protein in HepG2 cells. In contrast, these findings were not observed in Hep3B and Huh7 cells exposed to EPA. Most notably, EPA-induced apoptosis in HepG2 cells could be reduced almost completely by treatment with FasL antisense oligonucleotides. We conclude that EPA inhibits the growth of HepG2 cells and mediates its effect, at least in part, via the Fas-mediated apoptosis. It appears that the effects of EPA on hepatoma cells are determined by the status of p53 and that wild-type p53 is a prerequisite for the anticancer effect of EPA.     

Vaccinia virus-mediated expression of wild-type p53 suppresses glioma cell growth and induces apoptosis

The use of vaccinia virus vectors for cancer gene therapy may become a powerful method to achieve efficient anti-tumor effects. We used recombinant vaccinia virus expressing wild-type p53 (rVV-p53) to examine the biological effects of exogenous tumor suppressor p53 in human (U-373MG, U-87MG, LN-Z308) and rat glioma cells (9L, C6) in vitro. All glioma cell lines infected with rVV-p53 exhibited growth inhibition and underwent apoptosis as demonstrated by morphological studies using nuclear staining and flow cytometry. The key role of p53 in cell growth inhibition was confirmed as measured by colony forming efficiency. Growth inhibition and apoptosis were independent of the endogenous p53 status of the glioma cell lines.     

Overexpression of E2F1 in glioma-derived cell lines induces a p53-independent apoptosis that is further enhanced by ionizing radiation

Glioma cell lines show variable responses to radiation in a manner influenced by their p53 status. Irradiation of glioma cell lines does not generally induce apoptosis. When wild-type p53 is present, these cells undergo a G1 arrest that is closely associated with increased radiosensitivity as measured by clonogenic survival. Previously, others have shown that dysregulated overexpression of E2F1 induces apoptosis in cell lines with either functional or inactivated p53. We found that regardless of p53 status, apoptosis induced by overexpression of E2F1 in glioma cell lines was further enhanced by treatment with ionizing radiation. BAX induction did not follow E2F1 overexpression or irradiation in the glioma cell lines tested. Thus, the apoptotic response of glioma-derived cells to irradiation can be enhanced by E2F1 by a mechanism that does not involve the induction of BAX.     

HPV-16 E6 upregulation of DNMT1 through repression of tumor suppressor p53

The HPV-16 early proteins E6 and E7 are considered to function as oncoproteins in cervical cancer. DNA methyltransferase 1 (DNMT1) is one of the enzymes involved in epigenetic silencing of tumor suppressor genes. In the present study, the functional role and regulation of DNMT1 in HPV-16 E6 associated cervical cancer development were examined. Knockdown of E6 in HPV-16 positive human cervical carcinoma SiHa and CaSki cells led to the increase in p53, repression of DNMT1 protein and promoter activity. Moreover, p53 knockdown increased the DNMT1 protein as well as promoter activity, indicating that p53 may mediate E6 upregulation of DNMT1. In addition, E6 knockdown induced growth retardation in SiHa cells, and the effect was partially reverted by DNMT1 overexpression. The results suggest that HPV-16 E6 may act through p53/ DNMT1 to regulate the development of cervical cancer.     

Resveratrol induces apoptosis in breast cancer cells by E2F1-mediated up-regulation of ASPP1

Resveratrol is a natural polyphenolic compound with cancer chemopreventive activity. However, our understanding of the molecular mechanism responsible for resveratrol-induced apoptosis is still very limited. Here, we used MCF-7 and MDA-MB231 breast cancer cells as a model to demonstrate that resveratrol induced the expression of ASPP1, a new member of the ASPP (apoptosis stimulation protein of p53) family, which plays an important role in the regulation of apoptosis. Moreover, resveratrol enhanced apoptosis of MCF-7/ASPP1 cells, accompanied by higher expression of bax and p21. In contrast, siRNA-mediated knockdown of ASPP1 inhibited apoptosis in MB231 cells. Furthermore, we found that higher levels of ASPP1 were associated with adenovirus-mediated overexpression of E2F1 while siRNA-mediated E2F1 knockdown led to down-regulation of ASPP1. In conclusion, our results demonstrate that overexpression of ASPP1 rendered MCF-7 and MDA-MB231 breast cancer cells more sensitive to resveratrol-mediated apoptosis via the E2F pathway, thus suggesting that ASPP1 may represent a novel therapeutic target for resveratrol in human breast cancer.