Evidence of a functional role for p21WAF1/CIP1 down-regulation in synergistic antileukemic interactions between the histone deacetylase inhibitor sodium butyrate and flavopiridol

The functional significance of disruption of p21(WAF1/CIP1) induction by flavopiridol (FP) in human leukemia cells (Jurkat) exposed to the histone deacetylase (HDAC) inhibitor sodium butyrate (SB) was investigated. Coexposure of leukemic cells to FP blocked SB-mediated induction of p21(WAF1/CIP1) and resulted in a marked increase in mitochondrial injury, activation of procaspases-3 and -8, Bid cleavage, and PARP degradation. Enforced expression of p21(WAF1/CIP1) (i.e., in Jurkat cells inducibly expressing p21(WAF1/CIP1) under the control of a doxycycline-responsive promoter) partially but significantly reduced cytochrome c and apoptosis-inducing factor release, loss of mitochondrial membrane potential, caspase-3 and -8 activation, Bid cleavage, poly(ADP-ribose)polymerase (PARP) degradation, and apoptosis in response to SB/FP. Furthermore, increasing expression of p21(WAF1/CIP1) (i.e., by culturing cells in the presence of higher concentrations of doxycycline) rendered cells more resistant to SB/FP-mediated lethality. Enforced expression of p21(WAF1/CIP1) did not modify SB/FP-mediated JNK activation or generation of reactive oxygen species. Consistent with these results, Jurkat cells stably expressing a p21(WAF1/CIP1) nuclear localization mutant (p21DeltaNLS) were also resistant to SB/FP-mediated mitochondrial injury, activation of procaspases-3 and -8, PARP cleavage, and apoptosis. Finally, enforced expression of full-length or ectopic expression of DeltaNLS p21(WAF1/CIP1) increased the amount of p21(WAF1/CIP1) coimmunoprecipitating with procaspase-3. Together, these findings suggest that interruption of HDAC-mediated p21(WAF1/CIP1) induction by FP plays a significant functional role in potentiating apoptosis, possibly by preventing the formation of a procaspase-3/p21(WAF1/CIP1) complex.     

Modulation of mitogen-activated protein kinases by 6-nitro-7-hydroxycoumarin mediates apoptosis in renal carcinoma cells

6-Nitro-7-hydroxycoumarin has previously been shown to be a selective anti-proliferative agent capable of activating p38, stress-activated protein kinase (SAPK) and mitogen-activated protein (MAP) kinase in the human renal cell carcinoma cell line, A-498. Here, the role of p38 MAP kinase was further investigated in relation to its participation in 6-nitro-7-hydroxycoumarin induced apoptosis. 6-Nitro-7-hydroxycoumarin was shown to alter cell cycle progression, leading to the appearance of a sub-G(1) peak, containing hypodiploid DNA, accompanied by increases in both poly(ADP-ribose)polymerase cleavage and decreased expression of cyclin D1. Drug treatment also lead to a rise in the expression in the cyclin-dependent kinase inhibitor, p21(WAF1/CIP1), and the appearance of inter-nucleosomal DNA cleavage and morphological changes, consistent with apoptotic cell death. Using a p38 MAP kinase inhibitor, SB203580, caused expression of p21(WAF1/CIP1) to be suppressed and both cleaved poly(ADP-ribose)polymerase and the numbers of apoptotic cells were decreased. In summary, this study shows the participation of p38 MAP kinase in 6-nitro-7-hydroxycoumarin induced apoptosis of A-498 cells and suggests that targeting of p38 may represent a novel mechanism to inhibit renal cell carcinoma and that coumarin type drugs require further investigation as potential anticancer agents directed against renal cell carcinoma.     

新型组蛋白去乙酰化酶抑制剂致U251细胞周期阻滞、凋亡及其机制的研究

目的探讨新型组蛋白去乙酰化酶抑制剂(HDACi)2,2,3,3-四甲基环丙酰硫脲(TCCT)诱导人脑胶质瘤U251细胞周期阻滞、凋亡及其作用机制。方法以不同药物浓度与U251细胞共同培养48 h后,采用MTT法检测药物作用48 h后肿瘤细胞的增殖。药物作用24 h后,采用RT-PCR检测肿瘤细胞的p21WAF1/CIP1与Cyclin D1 mRNA的表达,Western blot检测HDAC3、HDAC4、Cyclin D1与p21WAF1/CIP1蛋白的表达,PI单染法分析细胞周期,Annexin V-PI双染法检测肿瘤细胞凋亡。结果 TCCT对U251细胞增殖具有明显抑制作用,药物干预48 h时的IC50为(0.461±0.108)mmol·L-1,并呈现剂量依赖性。TCCT药物干预24 h后U251细胞p21WAF1/CIP1mRNA表达上调,Cyclin D1 mRNA下调;组蛋白去乙酰化酶3(HDAC3)和组蛋白去乙酰化酶4(HDAC4)表达下调,Cyc-lin D1蛋白表达弱下调,p21WAF1/CIP1蛋白表达上调;S期细胞比例明显提高(P<0.05);细胞凋亡率明显增高(P<0.01)。结论 TCCT对U251细胞增殖有明显的抑制作用,引起肿瘤细胞S期阻滞和凋亡。其作用机制可能与其下调HDAC3、HDAC4表达,促进组蛋白乙酰化,而影响p21WAF1/CIP1和Cyclin D1的基因、蛋白的表达有关。     

Cytoprotective effects of hematopoietic growth factors on primary human acute myeloid leukemias are not mediated through changes in BCL-2, bax, or p21WAF1/CIP1

BACKGROUND: Hematopoietic growth factors (HGF) can suppress chemotherapy-induced programmed cell death (apoptosis) in hematopoietic cells. Although HGF can modulate the expression of apoptosis-regulatory genes, including bcl-2, bax, and p21WAF1/CIP1 in cell lines, few data address whether HGF regulate the expression of these proteins in primary acute myeloid leukemia (AML). MATERIALS AND METHODS: We evaluated the expression of bcl-2, bax, and p21WAF1/CIP1 in primary samples from patients with AML in the presence and absence of HGF. The potential association of HGF-induced changes in the levels of these proteins with inhibition of chemotherapy-induced apoptosis was further investigated. RESULTS: While a combination of steel factor (SF) and PIXY321 inhibited etoposide-induced apoptosis in 8/11 primary AML samples studied, Bcl-2 and bax protein levels were unaffected by exposure to HGF and/or etoposide. In contrast, HGF enhanced basal and etoposide-induced p21WAF1/CIP1 protein levels in 9/11 and 7/11 of the cases, respectively. In several cases, inhibition of apoptosis by HGF was seen without up-regulation of p21WAF1/CIP1 levels, suggesting that modulation of p21WAF1/CIP1 is not required for HGF-mediated inhibition of apoptosis. CONCLUSIONS: These data indicate that HGF-mediated inhibition of chemotherapy-induced apoptosis in primary AML samples is not mediated through changes in Bcl-2, bax, and p21WAF1/CIP1 protein levels.     

Induction of apoptosis in human breast adenocarcinoma MCF-7 cells by pterocarnin A from the bark of Pterocarya stenoptera via the Fas-mediated pathway

Pterocarnin A, isolated from the bark of Pterocarya stenoptera (Juylandaceae), was investigated for its antiproliferative activity in human breast adenocarcinoma MCF-7 cells. To identify the anticancer mechanism of pterocarnin A, we assayed its effects on apoptosis, cell cycle distribution, and levels of p53, p21/WAF1, Fas/APO-1 receptor and Fas ligand. The results showed that pterocarnin A induced apoptosis of MCF-7 cells without mediation of p53 and p21/WAF1. We suggest that the Fas/Fas ligand apoptotic system is the main pathway of pterocarnin A-mediated apoptosis of MCF-7 cells. Our study reports here for the first time that the activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of pterocarnin A in MCF-7 cells.     

LL-202, a newly synthesized flavonoid,inhibits tumor growth via inducing G2/M phase arrest and cell apoptosis in MCF-7 human breast cancer cells in vitro and in vivo

We recently established that LL-202, a newly synthesized flavonoid, exhibited obvious anticancer effects against human breast cells in vivo and in vitro. The underlying mechanism of its anticancer activity remains to be elucidated. In this study, we demonstrated that LL-202 inhibited the growth and proliferation of human breast cancer MCF-7 cells in a concentration and time-dependent manner. We reported that LL-202 induced both mitochondrial- and death-receptor-mediated apoptosis, which were characterized by the dissipation of mitochondrial membrane potential (ΔΨ_m), cytochrome c (Cyt c) release from mitochondria to cytosol, the activation of several caspases and induction of poly (ADP-ribose) polymerase (PARP) and Bid cleavage. N-acetylcysteine (NAC), a general ROS scavenger, partly blocked the LL-202-induced ROS levels and apoptosis. In addition, LL-202 induced arrest in cell cycle progression at G2/M phase in MCF-7 cells. After the treatment with LL-202, the expression of cell cycle-related proteins, such as cyclin B1, cyclin A, and p-CDK1 (Thr161) were down-regulated, whereas the expression of p21^WAF1/Cip1 and p-CDK1 (Thr14/Tyr15) were up-regulated. Finally, in vivo studies, LL-202 significantly suppressed the growth of MCF-7 breast cancer xenograft tumors in a dose-dependent manner with low systemic toxicity. In conclusion, the results showed that LL-202 had significant anticancer effects against human breast cells via the induction of apoptosis and G2/M phase arrest and it may be a novel anticancer agent for treatment of breast cancer.     

Peripheral-type benzodiazepine receptor overexpression and knockdown in human breast cancer cells indicate its prominent role in tumor cell proliferation

The peripheral-type benzodiazepine receptor (PBR), an 18-kDa high affinity drug and cholesterol binding protein, is expressed at high levels in various cancers. Its expression is positively correlated with aggressive metastatic behavior in human breast cancer cells. To determine the role of PBR in tumor progression, two human mammary carcinoma cell lines were utilized: the non-aggressive MCF-7 cell line, which expresses extremely low PBR levels, and the highly aggressive MDA-MB-231 cell line, which has much higher PBR levels. We have generated stably transfected lines of the tetracycline-repressible MCF-7 cell line (MCF-7 Tet-Off) with inducible human PBR cDNA. Induction of PBR expression in MCF-7 Tet-Off cells increased PBR ligand binding and cell proliferation. Transfection of MDA-MB-231 cells with multiple siRNAs complementary to PBR (PBR-siRNAs) led to different levels of PBR mRNA knockdown. Lentiviral-mediated PBR RNA interference in MDA-MB-231 cells decreased PBR levels by 50%. Decreased PBR expression was associated with cell cycle arrest at G2 phase, decreased cell proliferation, and significant increases in the protein levels of the cyclin-dependent kinase inhibitor p21(WAF/CIP1). These changes were accompanied by p53 activation seen as increased p53 phosphorylation (Ser15). In parallel, increased proteolytic activation of caspase-3 was also observed. Taken together these results suggest that PBR protein expression is directly involved in regulating cell survival and proliferation in human breast cancer cells by influencing signaling mechanisms involved in cell cycle control and apoptosis.     

Norsolorinic acid from Aspergillus nidulans inhibits the proliferation of human breast adenocarcinoma MCF-7 cells via Fas-mediated pathway

Norsolorinic acid, isolated from the Aspergillus nidulans, was investigated for its antiproliferative activity in human breast adenocarcinoma MCF-7 cells. To identity the anticancer mechanism of norsolorinic acid, we assayed its effect on apoptosis, cell cycle distribution, and levels of p53, p21/WAF1, Fas/APO-1 receptor and Fas ligand. The results showed that norsolorinic acid induced apoptosis of MCF-7 cells without mediation of p53 and p21/WAF1. We suggest that Fas/Fas ligand apoptotic system is the main pathway of norsolorinic acid-mediated apoptosis of MCF-7 cells. Our study reports here for the first time that the activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of norsolorinic acid in MCF-7 cells.     

Induction of apoptosis in human breast adenocarcinoma MCF-7 cells by prodelphinidin B-2 3,3'-di-O-gallate from Myrica rubra via Fas-mediated pathway

Myrica rubra Sieb et Zucc. (Myricaceae) is well known as a rich source of tannins. Prodelphinidin B-2 3,3'-di-O-gallate (PB233'OG) is a proanthocyanidin gallate that has been reported to exhibit antioxidant and antiviral activity. In this study, we evaluated the anti-proliferative activity of PB233'OG isolated from the bark of M. rubra in human breast adenocarcinoma MCF-7 cells. To identity the anti-cancer mechanism of PB233'OG, we assayed its effect on apoptosis, cell cycle distribution, and levels of p53, p21/WAF1, Fas/APO-1 receptor and Fas ligand. The results showed that PB233'OG induced apoptosis of MCF-7 cells without mediation of p53 and p21/WAF1. We suggest that Fas/Fas ligand apoptotic system is the main pathway of PB233'OG-mediated apoptosis of MCF-7 cells. Our study reports here for the first time that the activity of the Fas/Fas ligand apoptotic system may participate in the anti-proliferative activity of PB233'OG in MCF-7 cells.     

Casuarinin from the bark of Terminalia arjuna induces apoptosis and cell cycle arrest in human breast adenocarcinoma MCF-7 cells

Casuarinin, a hydrolyzable tannin isolated from the bark of Terminalia arjuna L. (Combretaceae), was investigated for its antiproliferative activity in human breast adenocarcinoma MCF-7 cells. The results showed that casuarinin inhibited the proliferation of MCF-7 by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. An enzyme-linked immunosorbent assay showed that casuarinin increased the expression of p21/WAF1 concomitantly as the MCF-7 cells underwent G0/G1 arrest. An enhancement in Fas/APO-1 and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by casuarinin. Our study reports here for the first time that the induction of p21/WAF1 and the activity of Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of casuarinin in MCF-7 cells.     

The anticancer agent prodigiosin induces p21WAF1/CIP1 expression via transforming growth factor-beta receptor pathway

The anticancer agent prodigiosin has been shown to act as an efficient immunosuppressant, eliciting cell cycle arrest at non-cytotoxic concentrations, and potent proapoptotic and antimetastatic effects at higher concentrations. Gene expression profiling of MCF-7 cells after treatment with a non-cytotoxic concentration of prodigiosin showed that expression of the p21WAF1/CIP1 gene, a negative cell cycle regulator was induced. In this study, we show that prodigiosin induces p21 expression leading to cell cycle blockade. Subsequently, we attempted to elucidate the molecular mechanisms involved in prodigiosin-mediated p21 gene expression. We demonstrate that prodigiosin induces p21 in a p53-independent manner as prodigiosin induced p21 in cells with both mutated and dominant negative p53. Conversely, the transforming growth factor-beta (TGF-beta) pathway has been found to be necessary for p21 induction. Prodigiosin-mediated p21 expression was blocked by SB431542, a TGF-beta receptor inhibitor. Nevertheless, this pathway alone is not enough to induce p21 expression. The TGF-beta family member (nonsteroidal anti-inflammatory drug)-activated gene 1/growth differentiation factor 15 (NAG-1) may activate this pathway, as it has previously been suggested to signal through the TGF-beta pathway and is overexpressed in response to prodigiosin treatment. We show that NAG-1 colocalizes with TGF-beta receptor type I, suggesting a possible interaction between them. Taken together, these results suggest the TGF-beta pathway is required for induction of p21 expression after prodigiosin treatment of MCF-7 cells.     

Putranjivain A from Euphorbia jolkini inhibits proliferation of human breast adenocarcinoma MCF-7 cells via blocking cell cycle progression and inducing apoptosis

Putranjivain A, isolated from the whole plant of Euphorbia jolkini Bioss (Euphorbiaceae), was investigated for its antiproliferative activity in human breast adenocarcinoma MCF-7 cells. The results showed that putranjivain A inhibited the proliferation of MCF-7 by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. Enzyme-linked immunosorbent assay showed that putranjivain A increased the expression of p21/WAF1 concomitantly as MCF-7 cell underwent G0/G1 arrest. An enhancement in Fas/APO-1 and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by putranjivain A. Our study reports here for the first time that the induction of p21/WAF1 and the activity of Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of putranjivain A in MCF-7 cells.     

Involvement of reactive oxygen species/c-Jun NH(2)-terminal kinase pathway in kotomolide A induces apoptosis in human breast cancer cells

The anticancer effects of kotomolide A (KTA), a new butanolide constituent isolated from the leaves of Cinnamomum kotoense (Lauraceae), on the two human breast cancer cell lines MCF-7 and MDA-MB-231, were first investigated in our study. KTA exhibited selectively antiproliferative effects in cancer cell lines without showing any toxicity in normal mammary epithelial cells. Treatment of cancer cells with KTA to trigger G2/M phase arrest was associated with increased p21/WAF1 levels and reduced amounts of cyclin A, cyclin B1, cdc2 and cdc25C. KTA induced cancer cell death treatment by triggering mitochondrial and death receptor 5 (DR5) apoptotic pathways, but did not act on the Fas receptor. Exposure of MCF-7 and MDA-MB-231 cells to KTA resulted in cellular glutathione reduction and ROS generation, accompanied by JNK activation and apoptosis. Both antioxidants, NAC and catalase, significantly decreased apoptosis by inhibiting the phosphorylation of JNK and subsequently triggering DR5 cell death pathways. The reduction of JNK expression by siRNA decreased KTA-mediated Bim cleavage, DR5 upregulation and apoptosis. Furthermore, daily KTA i.p. injections in nude mice with MDA-MB-231 s.c. tumors resulted in a 50% decrease of mean tumor volume, compared with vehicle-treated controls. Taken together, the data show that cell death of breast cancer cells in response to KTA is dependent upon ROS generation and JNK activation, triggering intrinsic and extrinsic apoptotic pathways. The ROS/JNK pathway could be a useful target for novel approaches in breast cancer chemotherapy.     

椎茸的乙酸乙脂提取物诱导人乳腺癌MCF-7细胞凋亡的作用

目的观察椎茸的乙酸乙脂提取物对体外培养的人乳腺癌MCF7细胞株诱导凋亡的作用。方法MTT法测定提取物对MCF7细胞生长的影响;AnnexinVFITC和PI染色,采用FCM测定MCF7细胞的凋亡指数;采用FCM分析细胞周期的影响;Westernblot法分析CyclinD1、Cdk4、Bax和p21WAE1/CIP1表达。结果椎茸的乙酸乙脂提取物可以剂量依赖性的抑制乳腺癌细胞株MCF7的生长,使MCF7细胞生长停滞在G0/G1期而诱导MCF7细胞凋亡;通过Bax和p21WAE1/CIP1的表达和降低CyclinD1、Cdk4的表达而诱导MCF7细胞的凋亡。结论椎茸的乙酸乙脂提取物通过诱导细胞凋亡而产生抗乳腺癌的活性。     

A phenylacetate derivative,SCK6, inhibits cell proliferation via G1 cell cycle arrest and apoptosis

Phenylacetate is a differentiation agent and has anticancer activity with relatively low toxicity. In the present study, we examined the anticancer effect of six synthetic phenylacetate derivatives in human lung cancer cells in our search for more effective phenylacetate analogous. Results showed that the antiproliferative effects of these synthetic compounds were stronger than those of phenylacetate, and that N-butyl-2-(2-fluorolphenyl)acetamide (SCK6) is the most potent compound. To address the mechanism of the antiproliferative effect of SCK6, cell cycle analysis was performed. Result showed that SCK6 (1 mM) induced G(1) arrest in CH27 cells. Western blot analysis of G(1) phase regulatory proteins demonstrated that the protein levels of cyclin-dependent kinase 2 (Cdk2), Cdk4, Cyclin E and Cyclin D3 were decreased after treatment with SCK6 but not those of Cdk6, Cyclin D1 and D2. In contrast, SCK6 increased the protein levels of p53 and p21(CIP1/WAF1). Data from in situ terminal transferase-mediated dUTP-fluorescensin nick end-labeling (TUNEL) assay and DNA fragmentation analysis demonstrated that SCK6 induced apoptotic cell death in CH27 cells. This SCK6-induced apoptosis was accompanied by a downregulation of Bcl-2 protein and activation of the caspase-9 cascade. Overexpression of Bcl-2 by adeno-Bcl-2 vector infection significantly inhibited SCK6-induced apoptosis. Moreover, treatment with caspase inhibitors also markedly reduced cell death induced by SCK6. Taken together, these results suggest that downregulation of G(1)-associated Cdks and cyclins and upregulation of p53 and p21(CIP1/WAF1) may contribute to SCK6-mediated G(1)-phase arrest. Furthermore, the decrease in Bcl-2 and the activation of caspase-9/caspase-3 may be the effector mechanism through which SCK6 induces apoptosis.