Naringenin inhibits migration,invasion, induces apoptosis in human lung cancer cells and arrests tumour progression in vitro

Lung cancer is one of the major cause for high-death rate all over the world, due to increased metastasize and difficulties in diagnosis. Naringenin is naturally occurring flavonoid found in various fruits including tomatoes, citrus fruit and figs. Naringenin is known to have several therapeutic effects including anti-atherogenic, antimicrobial, anti-inflammatory, hepatoprotective, anticancer and anti-mutagenic. The present study was aimed to analyse the naringenin induced anti-proliferative and apoptosis effects in human lung cancer cells. Cells were treated with various concentrations of naringenin (10, 100 & 200 µmol/L) for 48 hours. Cisplatin (20 µg/mL) was used as positive control. Cell viability, apoptosis, migration and mRNA, and protein expression of caspase-3, matrixmetallo proteinases-2 (MMP-2) and MMP-9 were determined. The cell viability was 93.7 ± 7.5, 51.4 ± 4.4 and 32.1 ± 2.1 at 10, 100 and 200 µmol/L of naringenin respectively. Naringenin significantly increased apoptotic cells. The 100 and 200 µmol/L of naringenin significantly suppressed the larger wounds of cultured human cancer cells compared with the untreated lung cancer cells. Naringenin increased d the expression of caspase-3 and reduced the expression of MMP-2 and MMP-9. Taking all these data together, it is suggested that the naringenin was effective against human lung cancer proliferation, migration and metastasis.     

Nuclear BAG-1 expression inhibits apoptosis in colorectal adenoma-derived epithelial cells

BAG-1 is an anti-apoptotic protein that is frequently deregulated in a variety of malignancies including colorectal cancer. There are three isoforms: BAG-1L is located in the nucleus, BAG-1M and BAG-1S are located both in the nucleus and the cytoplasm. In colon cancer, the expression of nuclear BAG-1 is associated with poorer prognosis and is potentially a useful predictive factor for distant metastasis. However, the function of BAG-1 in colonic epithelial cells has not been studied. Having previously shown a predominant nuclear localisation of BAG-1 in adenoma-derived cell lines,¹ we wanted to determine the function of nuclear BAG-1 in these non-tumourigenic cells, to identify whether nuclear BAG-1 was implicated in tumour progression in the colon. In the current report we established that nuclear BAG-1 inhibits apoptosis in a colorectal adenoma-derived cell line. We demonstrate that apoptosis induced by -radiation or the vitamin D analogue EB1089 in the non-tumourigenic human colorectal adenoma-derived S/RG/C2 cell line, was preceded by a decrease in nuclear and an increase in cytoplasmic BAG-1 expression. This change in subcellular localisation of BAG-1 was due to the redistribution of the BAG-1M isoform. In addition, we have shown that the maintenance of high nuclear BAG-1 through enforced expression of the nuclear localised BAG-1L isoform enhanced cellular survival after -radiation or exposure to EB1089. Furthermore the expression of cytoplasmic BAG-1S isoform fused with a nuclear localisation signal protected against -radiation induced apoptosis. This demonstrates that nuclear localisation of the BAG-1 protein confers a survival advantage in colorectal adenoma-derived cells and that nuclear BAG-1 could potentially be an important survival factor in colorectal carcinogenesis.     

Insulin-like growth factors induce apoptosis as well as proliferation in LIM 1215 colon cancer cells

The insulin-like growth factor (IGF) system plays an important role in cell proliferation and survival. However, more recently, a small number of studies have shown that IGFs induce apoptosis in some cells. Our initial studies showed this occurred in LIM 1215 colon cancer cells but not RD rhabdomyosarcoma cells. IGFs induced both proliferation and apoptosis in LIM 1215 cells, and the induction of apoptosis was dose-dependent. [R54, R55]IGF-II, which binds to the IGF-I receptor with normal affinity but does not bind to the IGF-II receptor, induced apoptosis to the same extent as IGF-II, whereas [L27]IGF-II, which binds to the IGF-I receptor with 1000-fold reduced affinity, had no effect on apoptosis. These results suggest that the IGF-I receptor is involved in induction of apoptosis. Western blot analyses demonstrated that Akt and Erk1/2 were constitutively activated in RD cells. In contrast, phosphorylation of Akt and Erk1/2 were transient and basal expression of Akt protein was lower in LIM 1215 cells. Analysis of apoptosis-related proteins showed that IGFs decreased pro-caspase-3 levels and increased expression of pro-apoptotic Bad in LIM 1215 cells. IGFs co-activate proliferative and apoptotic pathways in LIM 1215 cells, which may contribute to increased cell turnover. Since high turnover correlates with poor prognosis in colorectal cancer, this study provides further evidence for the role of the IGF system in its progression.     

Combination of taxol and Bcl‐2 siRNA induces apoptosis in human glioblastoma cells and inhibits invasion,angiogenesis and tumour growth

Taxol is a powerful chemotherapeutic agent that binds to microtubules to prevent tumour cell division. However, a traditional high dose of taxol may also induce apoptosis in normal cells. The anti‐apoptotic molecule Bcl‐2 is up‐regulated in tumour cells to prevent apoptosis. We designed this study to determine whether use of a low dose of taxol and anti‐apoptotic Bcl‐2 gene silencing would effectively induce apoptosis in human glioblastoma U251MG cells and also inhibit invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. We treated the cells with either 100 nM taxol or transfected with a plasmid vector expressing Bcl‐2 siRNA or both agents together for 72 h. Knockdown of Bcl‐2 potentiated efficacy of taxol for cell death. Fluorescence‐activated cell sorting analysis, double immunofluorescent staining and TUNEL assay demonstrated apoptosis in about 70% of the cells after treatment with the combination of taxol and Bcl‐2 siRNA. In vitro Matrigel invasion assay demonstrated dramatic decrease in glioblastoma cell invasion and in vivo angiogenesis assay showed complete inhibition of neovascularization in athymic nude mice after treatment with the combination. Further, treatment with the combination of taxol and Bcl‐2 siRNA caused suppression of intracranial tumour growth and subcutaneous solid tumour development. In conclusion, our results indicate that the combination of taxol and Bcl‐2 siRNA effectively induces apoptosis and inhibits glioblastoma cell invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. Therefore, the combination of a low dose of taxol and Bcl‐2 siRNA is a promising therapeutic strategy for controlling the aggressive growth of human glioblastoma.     

Vector-based miR-15a/16-1 plasmid inhibits colon cancer growth in vivo

miR-15 (microRNA 15) and miR-16 are frequently deleted or down-regulated in many cancer cell lines and various tumour tissues, suggesting that miR-15a/16-1 plays important roles in tumour progression and might be a method for cancer treatment. We have developed a vector-based plasmid to explore the anti-tumour efficacy of miR-15a/16-1 in colon cancer in vivo. It is proposed that miR-15a and miR-16-1 target cyclin B1 (CCNB1), which associates with several tumorigenic features such as survival and proliferation. The levels of miR-15a and miR-16-1 in colon cancer cells were inversely correlated with CCNB1 expression, and there was consensus between miR-15a/16-1 and CCNB1 mRNA sequences by analysing homology. Vector-based miR-15a/16-1 expression plasmid was constructed and transfected into HCT 116 and SW620 colon cancer cells in vitro. The effects produced on cell viability and angiogenesis were analysed using flow cytometric analysis, colony formation analysis and tube formation analysis. CCNB1 expression down-regulation was checked by Western blotting. Systemic delivery of miR-15a/16-1 plasmids encapsulated in cationic liposome led to a significant inhibition of subcutaneous tumour growth and angiogenesis in tumour tissues, whereas no effects were observed with liposome carrying the non-specific plasmid. In summary, miR-15a/16-1 has been applied in colon cancer treatment in vivo, and resulted in effective colon tumour xenografts growth arrest and angiogenesis decrease. These findings suggest that systemic delivery of vector-based miR-15a/16-1 expression plasmid can be an approach to colon cancer therapy.     

Mechanism of taurine-induced apoptosis in human colon cancer cells

Taurine （Tau） has been shown to possess cancer therapeutic effect through induction of apoptosis, while the underlying molecular mechanism of its anti-cancer effect is not well understood. PUMA （p53-upregulated modulator of apoptosis） plays an important role in the process of apoptosis induction in a variety of human tumor ceils in both p53- dependent and -independent manners. However, whether PUMA is involved in the process of Tau-induced apoptosis in cancer cells has not been well studied. In the present study, we treated human colorectal cancer cells HT-29 （mutant p53） and LoVo （wild-type p53） with different concentrations of Tau, which led to the repression of cell proliferation and induction of apoptosis in both cell fines. Meanwhile, we also observed the increased expression of PUMA and high Bax/Bcl-2 ratios. To determine the role of PUMA in Tau-induced apoptosis, we used small interfering RNA interference to suppress PUMA expression. As a result, apoptosis was decreased in response to Tau treatment. All these results indicated that PUMA plays a critical role in Tauinduced apoptosis pathway in human colorectal cancer ceils. Demonstration of the molecular mechanism involved in the anti-tumor effect of Tau may be useful in the therapeutic target selection for p53-deficient colorectal cancer.     

Agkistin-s, a disintegrin domain, inhibits angiogenesis and induces BAECs apoptosis

Previous work in our laboratory has shown agkistin, a snake venom metalloproteases (SVMPs) from the venom of Agkistrodon halys, possesses antiplatelet aggregation activity. In this study, we further examined the antiangiogenic activity of agkistin-s, the disintegrin domain of agkistin. Recombinant agkistin-s was produced in Escherichia coli by subcloning its cDNA into pET28a vector, and the effect of purified agkistin-s was evaluated. At the concentration of 0.5-1.5 microM, the recombinant agkistin-s exhibited inhibitory activities on the bovine aortic endothelial cells (BAECs) migration and proliferation in a dose-dependent manner. In addition, it exhibited an effective antiangiogenic effect when assayed by using the 10-day-old embryo chick CAM model and effectively inhibits the tube-like structure formation. Furthermore, it potently induced BAECs apoptosis as examined by flow cytometric assays.     

Midazolam induces cellular apoptosis in human cancer cells and inhibits tumor growth in xenograft mice

Midazolam is a widely used anesthetic of the benzodiazepine class that has shown cytotoxicity and apoptosisinducing activity in neuronal cells and lymphocytes. This study aims to evaluate the effect of midazolam on growth of K562 human leukemia cells and HT29 colon cancer cells. The in vivo effect of midazolam was investigated in BALB/c-nu mice bearing K562 and HT29 cells human tumor xenografts. The results show that midazolam decreased the viability of K562 and HT29 cells by inducing apoptosis and S phase cell-cycle arrest in a concentration-dependent manner. Midazolam activated caspase-9, capspase-3 and PARP indicating induction of the mitochondrial intrinsic pathway of apoptosis. Midazolam lowered mitochondrial membrane potential and increased apoptotic DNA fragmentation. Midazolam showed reactive oxygen species (ROS) scavenging activity through inhibition of NADPH oxidase 2 (Nox2) enzyme activity in K562 cells. Midazolam caused inhibition of pERK1/2 signaling which led to inhibition of the anti-apoptotic proteins Bcl-X_L and XIAP and phosphorylation activation of the pro-apoptotic protein Bid. Midazolam inhibited growth of HT29 tumors in xenograft mice. Collectively our results demonstrate that midazolam caused growth inhibition of cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis and inhibited HT29 tumor growth in xenograft mice. The mechanism underlying these effects of midazolam might be suppression of ROS production leading to modulation of apoptosis and growth regulatory proteins. These findings present possible clinical implications of midazolam as an anesthetic to relieve pain during in vivo anticancer drug delivery and to enhance anticancer efficacy through its ROS-scavenging and pro-apoptotic properties.     

Minerval induces apoptosis in Jurkat and other cancer cells

Minerval is an oleic acid synthetic analogue that impairs lung cancer (A549) cell proliferation upon modulation of the plasma membrane lipid structure and subsequent regulation of protein kinase C localization and activity. However, this mechanism does not fully explain the regression of tumours induced by this drug in animal models of cancer. Here we show that Minerval also induced apoptosis in Jurkat T‐lymphoblastic leukaemia and other cancer cells. Minerval inhibited proliferation of Jurkat cells, concomitant with a decrease of cyclin D3 and cdk2 (cyclin‐dependent kinase2). In addition, the changes that induced on Jurkat cell membrane organization caused clustering (capping) of the death receptor Fas (CD95), caspase‐8 activation and initiation of the extrinsic apoptosis pathway, which finally resulted in programmed cell death. The present results suggest that the intrinsic pathway (associated with caspase‐9 function) was activated downstream by caspase‐8. In a xenograft model of human leukaemia, Minerval also inhibited tumour progression and induced tumour cell death. Studies carried out in a wide variety of cancer cell types demonstrated that apoptosis was the main molecular mechanism triggered by Minerval. This is the first report on the pro‐apoptotic activity of Minerval, and in part explains the effectiveness of this non‐toxic anticancer drug and its wide spectrum against different types of cancer.     

Inhibition of NEDD4 inhibits cell growth and invasion and induces cell apoptosis in bladder cancer cells

The neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) plays a pivotal oncogenic role in various types of human cancers. However, the function of NEDD4 in bladder cancer has not been fully investigated. In the present study, we aim to explore whether NEDD4 governs cell proliferation, apoptosis, migration, and invasion in bladder cancer cells. Our results showed that downregulation of NEDD4 suppressed cell proliferation in bladder cancer cells. Moreover, we found that inhibition of NEDD4 significantly induced cell apoptosis. Furthermore, downregulation of NEDD4 retarded cell migration and invasion. Notably, overexpression of NEDD4 enhanced cell growth and inhibited apoptosis. Consistently, upregulation of NEDD4 promoted cell migration and invasion in bladder cancer cells. Mechanically, our Western blotting results revealed that downregulation of NEDD4 activated PTEN and inhibited Notch-1 expression, whereas upregulation of NEDD4 reduced PTEN level and increased Notch-1 level in bladder cancer cells. Our findings indicated that NEDD4 exerts its oncogenic function partly due to regulation of PTEN and Notch-1 in bladder cancer cells. These results further revealed that targeting NEDD4 could be a useful approach for the treatment of bladder cancer.     

Phloroglucinol derivative MCPP induces cell apoptosis in human colon cancer

This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (3,6-bis(3-chlorophenylacetyl)phloroglucinol; MCPP) in human colon cancer cells. MCPP induced cell death and antiproliferation in three human colon cancer, HCT-116, SW480, and Caco-2 cells, but not in primary human dermal fibroblast cells. MCPP-induced concentration-dependent apoptotic cell death in colon cancer cells was measured by fluorescence-activated cell sorter (FACS) analysis. Treatment of HCT-116 human colon cancer cells with MCPP was found to induce a number of signature endoplasmic reticulum (ER) stress markers; and up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein (GRP)-78, phosphorylation of eukaryotic initiation factor-2α (eIF-2α), suggesting the induction of ER stress. MCPP also increased GSK3α/β(Tyr270/216) phosphorylation and reduced GSK3α/β(Ser21/9) phosphorylation time-dependently. Transfection of cells with GRP78 or CHOP siRNA, or treatment of GSK3 inhibitor SB216163 reduced MCPP-mediated cell apoptosis. Treatment of MCPP also increased caspase-7, caspase-9, and caspase-3 activity. The inhibition of caspase activity by z-DEVE-FMK or z-VAD-FMK significantly reduced MCPP-induced apoptosis. Furthermore, treatment of GSK3 inhibitor SB216763 also dramatically reversed MCPP-induced GRP and CHOP up-regulation, and pro-caspase-3 and pro-caspase-9 degradation. Taken together, the present study provides evidences to support that GRP78 and CHOP expression, and GSK3α/β activation in mediating the MCPP-induced human colon cancer cell apoptosis.     

Betulinic acid induces apoptosis and inhibits metastasis of human colorectal cancer cells in vitro and in vivo

Betulinic acid (BA) is a pentacyclic triterpenoids extracted from birch with a wide range of biological properties. Recent studies have shown that BA has significant cytotoxicity to various types of human cancer cells, and shows potential in cancer treatment. However, the efficacy of BA on human colorectal cancer tumor cells is still unclear. The purpose of our study was to evaluate the anti-cancer activity of BA in human colorectal cancer cells in vitro and in vivo to investigate the possible mechanism. In this experiment, we found that BA inhibited colorectal cancer cell lines in vitro with a time-dependent and dose-dependent manner. Moreover, BA could induce cell apoptosis by upregulating expression of Bax and cleaved caspase-3 and downregulating protein of Bcl-2. BA could increase the production of reactive oxygen species and reduce mitochondrial membrane potential of cancer cell, suggesting that BA induced cancer cells apoptosis by mitochondrial mediated pathways. Furthermore, BA significantly inhibited the migration and invasion of colorectal cancer cells, reduced the expression of matrix metalloproteinase (MMPs) and increased the expression of MMPs inhibitor (TIMP-2). In addition, the growth of tumor was significantly suppressed by intraperitoneal administration of 20 mg/kg/day of BA in a xenograft tumor mouse model of HCT-116. Histopathological and immunohistochemical analysis showed that MMP-2+ cells and Ki-67+ cells were reduced and cleaved caspase-3+ cells were increased in tumor tissues of mice after BA administration. The results showed that BA not only promoted the apoptosis of colorectal cancer cells, but also inhibited the metastasis of cancer cells. Our results suggest that BA can be a potential natural drug to inhibit the growth and metastasis of colorectal cancer.     

黄精凝集素PCL-2诱导人前列腺癌LNCap细胞凋亡及机制研究

研究黄精凝集素PCL-2对人前列腺癌LNCap细胞生物活性和可能的抗肿瘤机制。从黄精药材中提取分离得到黄精凝集素PCL-2,体外培养LNCap细胞,采用WST-1和集落形成实验评价PCL-2对LNCap细胞的增殖作用;2,7-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针法检测LNCap细胞内ROS含量;qRT-PCR和Western blot法检测PCL-2对LNCap细胞中相关基因和蛋白表达的影响。研究结果显示PCL-2能显著抑制LNCap细胞的增殖,促进细胞中ROS生成;PCL-2通过上调LNCap细胞中Bax、Caspase-3及Caspase-9基因mRNA和蛋白表达并下调Bcl-2基因mRNA和蛋白表达水平诱导LNCap细胞凋亡。本研究结果表明PCL-2诱导LNCap细胞凋亡活性作用显著,可作为抑制前列腺癌的潜在药物。     

A ginger derivative,zingerone—a phenolic compound—induces ROS‐mediated apoptosis in colon cancer cells (HCT‐116)

Zingerone (ZO), an active phenolic agent derived from Zingiber officinale (Ginger), has many pharmacological properties such as antioxidant, antiangiogenic, and antitumor. However, its potential value in cancer and the mechanism by which ZO wields its therapeutic effects remain obscure. Therefore, in this current study, we explored the effects of ZO on suppressing cell proliferation and enhancing apoptosis in colon cancer cells (HCT116). Our results indicated that ZO significantly enhances the production of reactive oxygen species, lipid peroxidation (thiobarbituric acid reactive substance [TBARS]), and loss of cell viability; and reduces mitochondrial membrane potential and antioxidant levels (SOD, CAT, and GSH) in ZO‐treated HCT116 cells in a dose‐dependent (2.5, 5, and 10 µM) manner. Furthermore, ZO induces oxidative stress‐mediated apoptosis as evidenced by apoptotic morphological changes predicted by AO/EtBr, Hoechst staining and further confirmed by comet assay. Moreover, immunoblotting techniques showed that ZO treatment effectively enhances Bax, caspase‐9, and caspase‐3 expressions and decreases the expression of Bcl‐2 in colon cancer cells. Together, our results evidenced that the antitumor effects of ZO reduce cell proliferation and stimulate apoptosis through modulating pro‐ and antiapoptotic molecular events in HCT116 colon cancer cells. Therefore, based on our findings, ZO may be used as a therapeutic agent for the treatment of colon cancer.     

Serine/threonine kinase 31 promotes PDCD5-mediated apoptosis in p53-dependent human colon cancer cells

Although programed cell death 5 (PDCD5) is an important protein in p53-mediated proapoptotic signaling, very little is known about PDCD5-related cell death. In this study, we report that serine/threonine kinase 31 (STK31) interacts with PDCD5, which maintains the stability of PDCD5. STK31 overexpression significantly activated PDCD5 stabilization and p53-mediated apoptosis in response to etoposide (ET). However, STK31 knockdown did not enhance apoptosis by ET treatment. Moreover, when STK31 was depleted, PDCD5 inhibited the activation of the p53 signaling pathway with ET, indicating that the PDCD5–STK31 network has an essential role in p53 activation. Importantly, STK31 activated the p53 signaling pathway by genotoxic stress through positive regulation of PDCD5-mediated apoptosis. We thus demonstrated that overexpression of STK31 greatly inhibited tumorigenic growth and increased the chemosensitivity of HCT116 human colorectal carcinoma cells. Taken together, these findings demonstrate that the STK31–PDCD5 complex network regulates apoptosis of cancer cells, and STK31 is a positive apoptosis regulator that inhibits tumorigenesis of colon cancer cells by inducing PDCD5-mediated apoptosis in response to genotoxic stress.     

Inhibition of colon cancer growth and metastasis by NK4 gene repetitive delivery in mice

NK4, originally prepared as a competitive antagonist for hepatocyte growth factor (HGF), is a bifunctional molecule that acts as an HGF-antagonist and angiogenesis inhibitor. When the expression plasmid for NK4 gene was administered into mice by hydrodynamics-based delivery, the repetitive increase in the plasma NK4 protein level was achieved by repetitive administration of NK4 gene. Mice were subcutaneously implanted with colon cancer cells and weekly given with the NK4 plasmid. The repetitive delivery and expression of NK4 gene inhibited angiogenesis and invasiveness of colon cancer cells in subcutaneous tumor tissue and this was associated with suppression of primary tumor growth. By fifty days after tumor implantation, cancer cells naturally metastasized to the liver, whereas NK4 gene expression potently inhibited liver metastasis. Inhibition of the HGF-Met receptor pathway and tumor angiogenesis by NK4 gene expression has potential therapeutic value toward inhibition of invasion, growth, and metastasis of colon cancer.