Xanthones induce cell-cycle arrest and apoptosis in human colon cancer DLD-1 cells

We investigated the antiproliferative effects of four structurally similar prenylated xanthones, alpha-mangostin, beta-mangostin, gamma-mangostin, and methoxy-beta-mangostin, in human colon cancer DLD-1 cells. These xanthones differ in the number of hydroxyl and methoxy groups. Except for methoxy-beta-mangostin, the other three xanthones strongly inhibited cell growth at 20 microM and their antitumor efficacy was correlated with the number of hydroxyl groups. Hoechst 33342 nuclear staining and nucleosomal DNA-gel electrophoresis revealed that the antiproliferative effects of alpha- and gamma-mangostin, but not that of beta-mangostin, were associated with apoptosis. It was also shown that their antiproliferative effects were associated with cell-cycle arrest by affecting the expression of cyclins, cdc2, and p27; G1 arrest was by alpha-mangostin and beta-mangostin, and S arrest by gamma-mangostin. These findings provide a relevant basis for the development of xanthones as an agent for cancer prevention and combination therapy with anti-cancer drugs.     

Overexpression of Par-4 enhances thapsigargin-induced apoptosis via down-regulation of XIAP and inactivation of Akt in human renal cancer cells

The prostate-apoptosis-response-gene-4 (Par-4) protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor-mediated cell death pathways. We found that overexpressing Par-4 by stable transfection sensitizes Caki cells to induction of apoptosis by TRAIL and drugs that induce endoplasmic reticulum (ER) stress [thapsigargin (TG), tunicamycin (TU) and etoposide]. Ectopic expression of Par-4 is associated with decreased levels of XIAP protein in TG-treated cells, caused in part by XIAP protein instability and caspase activation. Levels of phospho-Akt are decreased in Caki/Par-4 cells to a significantly greater extent than in Caki/Vector cells by treatment with TG, and this is in turn associated with decreased levels of phospho-PDK1, the kinase upstream of Akt. In conclusion, we provide evidence that ectopic expression of Par-4 sensitizes Caki cells to TG and that XIAP protein instability and inactivation of Akt are important in cellular pathways affected by Par-4.     

Berberine enhances posttranslational protein stability of p21/cip1 in breast cancer cells via down-regulation of Akt

Berberine has shown anticancer properties and has potential for a chemopreventive and/or chemotherapeutic agent for breast cancer. Berberine showed cytotoxicity to breast cancer cells, with an increase in the levels of p21/cip1 and p27/kip1, cyclin-dependent kinase inhibitors (CDKI), but mechanisms involved in up-regulating these molecules are largely unknown. Herein, we studied the key regulatory mechanisms involved in berberine-mediated up-regulation of p21/cip1 and p27/kip1. Berberine treatment for 24 and 48 h decreased the number of cells by 44–84% (P?<?0.0001) and 38–78% (P?<?0.0001), and increased cell death by 12–17% (P?<?0.005) and 38–78% (P?<?0.0001) in MCF-7 and MDA-MB-231 cells, respectively. Cells were arrested in G1 phase by berberine which was accompanied with up-regulation of mRNA and protein level of both p21/cip1 and p27/kip1. Berberine decreased the expression of protein levels of cyclin D1, cyclin E, CDK2, CDK4, and CDK6 to cause G1 phase arrest. Berberine caused nuclear localization of p21/cip1 in both the cell lines. Our data for the first time showed that the post-translational stability of both the proteins was strongly increased by berberine as examined by cycloheximide chase assay. Inhibition of Akt was associated with berberine-mediated up-regulation of p21/cip1 and also led to a decrease in cell viability accompanied with significant G1 phase cell cycle arrest. Our study revealed that berberine not only up-regulates mRNA and protein levels of p21/cip1 and p27/kip1 but also increases their nuclear localization and post-translational protein stability. Further, Akt inhibition was found to mediate berberine-mediated up-regulation of p21/cip1 but not the p27/kip1.

     

Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress and leads to activation of the unfolded protein response, which reduces the stress and promotes cell survival at the early stage of stress, or triggers cell death and apoptosis when homeostasis is not restored under prolonged ER stress. Here, we report that Cab45S, a member of the CREC family, inhibits ER stress-induced apoptosis. Depletion of Cab45S increases inositol-requiring kinase 1 (IRE1) activity, thus producing more spliced forms of X-box-binding protein 1 mRNA at the early stage of stress and leads to phosphorylation of c-Jun N-terminal kinase, which finally induces apoptosis. Furthermore, we find that Cab45S specifically interacts with 78-kDa glucose-regulated protein/immunoglobulin heavy chain binding protein (GRP78/BiP) on its nucleotide-binding domain. Cab45S enhances GRP78/BiP protein level and stabilizes the interaction of GRP78/BiP with IRE1 to inhibit ER stress-induced IRE1 activation and apoptosis. Together, Cab45S, a novel regulator of GRP78/BiP, suppresses ER stress-induced IRE1 activation and apoptosis by binding to and elevating GRP78/BiP, and has a role in the inhibition of ER stress-induced apoptosis.     

Increased sensitivity of human colon cancer cells to DNA cross-linking agents after GRP78 up-regulation

We have shown earlier that pre-treatment of V79 Chinese hamster cells with 6-aminonicotinamide (6-AN) or 2-deoxyglucose (2-dG) results in over-expression of the Mr 78,000 glucose-regulated stress protein (GRP78) and the subsequent development of resistance to inhibitors of topoisomerase II. These phenomena also occur in V79-derived cell lines that are deficient in poly(ADP-ribose) (p(ADPR)) metabolism. In contrast, over-expression of GRP78 under the conditions outlined above is found to be associated with hypersensitivity to several clinically-relevant DNA cross-linking agents, namely, 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, and melphalan. We have also previously shown that pre-treatment with 6-AN, an inhibitor of p(ADPR) metabolism, causes an increase in the life span in BCNU-treated mice bearing L1210 tumors. These observations prompted us to examine whether 6-AN pre-treatment can result in the over-expression of GRP78 in human colon cancer cell lines and, if so, whether this increase is associated with sensitization to DNA cross-linking agents outlined above. Following treatment of three colon cancer cell lines, HCT116, SW480, and VACO-8, for 48 h with 0.1 mM 6-AN, cytosolic GRP78 levels were elevated approximately 4.2 times, 8 times, and 2.5 times for each cell line respectively, as measured by Western immunoblotting. To determine sensitivity after GRP78 up-regulation, the cells were washed and grown for 412 h in growth medium devoid of 6-AN, before being treated with DNA cross-linking agents. The 412 h time period allowed p(ADPR) metabolism to return to normal while GRP78 levels remained elevated, thus allowing us to associate GRP78 over-expression with sensitivity to those agents. After treating cells for 1 h with BCNU, cisplatin, or melphalan, cell sensitivity was determined by clonogenic survival assay or a fluorescence-based cytotoxicity assay. Based on changes in IC50 values, 6-AN caused an increase in sensitivity for HCT116, SW480, and VACO-8 cells of 1.5, 2.3, and 1.0 times, respectively, for BCNU, 4.8, 3.8, and 2.6 for cisplatin, and 6.4, 3.7, and 2.2 times for melphalan. Thus, our results show that over-expression of GRP78 in human tumor cell lines is associated with increased sensitivity to clinically useful chemotherapy agents. This sensitization occurred in three different tumor cell lines, each bearing a separate genetic defect associated with altered sensitivity.     

Arsenic trioxide-induced apoptosis through oxidative stress in cells of colon cancer cell lines

Exposure of three colon cancer cell lines, SW480, DLD-1, and COLO201, to arsenic trioxide in the medium induced a marked concentration-dependent suppression of cell growth. The intracellular contents of reduced glutathione (GSH) in these cell lines tended to be inversely correlated with the sensitivity of the cells to arsenic trioxide. Among the cell lines, SW480 cells underwent apoptosis at the low arsenic trioxide concentration of 2 microM, which was prevented by pretreatment of the cells with N-acetylcysteine and was enhanced by buthionine sulfoximine. The production of reactive oxygen intermediates which were examined by 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA), increased with time after treatment with arsenic trioxide. The apoptosis was executed by the activation of caspase 3, which was shown by Western blot, enzymatic activity, and apoptosis inhibition assay. The mitochondrial membrane potential of adherent apoptotic SW480 cells and the cells from intermediate layer separated by density gradient centrifugation, both of which showed the active form of caspase 3 by Western blot analysis, was not lost. The overexpression of Bcl-2 protein in SW480 cells could not prevent the apoptosis induced by the treatment with arsenic trioxide. All these findings indicate that arsenic trioxide-induced apoptosis in SW480 cells is executed by the activation of caspase 3 without mediating by mitochondria under the overproduction of reactive oxygen species.     

Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells

The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min–2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA₂. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.     

Heme oxygenase-1 inhibits apoptosis in Caco-2 cells via activation of Akt pathway

Heme oxygenase-1 can play a protective role against cellular stress. In colon cancer cells, these effects would be relevant to oncogenesis and resistance to chemotherapy. The aim of the study was to examine the effects of heme oxygenase-1 induction on cell survival in a human colon cancer cell line, Caco-2. Serum deprivation induced apoptosis, reduced Akt and p38 phosphorylation, and increased p21(Cip/WAF1) levels. Heme oxygenase-1 induction by treatment with cobalt protoporphyrin IX resulted in resistance to apoptosis, activation of Akt, reduction in p21(Cip/WAF1) levels and modification of bcl2/bax ratio towards survival. Indomethacin reduced apoptosis but in contrast to heme oxygenase-1, arrested cells in G0/G1. Apoptosis was also inhibited by the heme oxygenase metabolites bilirubin and biliverdin but the CO donor tricarbonyldichlororuthenium(II) dimer did not exert significant effects. Protection against apoptosis in cells treated with cobalt protoporphyrin IX was reverted by incubation with heme oxygenase-1 small interfering RNA. This study shows an antiapoptotic effect of heme oxygenase-1 in colon cancer cells which could be mediated by the formation of bilirubin and biliverdin. Our results support an antiapoptotic role for HO-1 in these cells and provide a mechanism by which overexpression of HO-1 may promote tumor resistance to stress in conditions of limited nutrient supply. We have extended these observations by demonstrating that these effects are independent of p38 but are mediated via Akt pathway.     

Induction of apoptosis in DLD-1 human colon cancer cells by peridinin isolated from the dinoflagellate, Heterocapsa triquetra

Peridinin, which is uniquely present in dinoflagellates, is one of the most abundant carotenoids found in nature. We evaluated the apoptotic effect of peridinin on DLD-1 human colorectal cancer cells. Peridinin significantly reduced the cell viability in a dose-dependent manner (0-20 microM) and induced apoptosis by activating both caspase-8 and caspase-9. Our findings could be important for the high-performance utilization of marine bioproducts.     

FLASH knockdown sensitizes cells to Fas-mediated apoptosis via down-regulation of the anti-apoptotic proteins, MCL-1 and Cflip short

FLASH (FLICE-associated huge protein or CASP8AP2) is a large multifunctional protein that is involved in many cellular processes associated with cell death and survival. It has been reported to promote apoptosis, but we show here that depletion of FLASH in HT1080 cells by siRNA interference can also accelerate the process. As shown previously, depletion of FLASH halts growth by down-regulating histone biosynthesis and arrests the cell cycle in S-phase. FLASH knockdown followed by stimulating the cells with Fas ligand or anti-Fas antibodies was found to be associated with a more rapid cleavage of PARP, accelerated activation of caspase-8 and the executioner caspase-3 and rapid progression to cellular disintegration. As is the case for most anti-apoptotic proteins, FLASH was degraded soon after the onset of apoptosis. Depletion of FLASH also resulted in the reduced intracellular levels of the anti-apoptotic proteins, MCL-1 and the short isoform of cFLIP. FLASH knockdown in HT1080 mutant cells defective in p53 did not significantly accelerate Fas mediated apoptosis indicating that the effect was dependent on functional p53. Collectively, these results suggest that under some circumstances, FLASH suppresses apoptosis.     

Matrine inhibits proliferation and induces apoptosis of human colon cancer LoVo cells by inactivating Akt pathway

The present study has investigated the anti-tumor activity and the underlying mechanisms of matrine on human colon cancer LoVo cells. Matrine inhibited the proliferation of the cells in dose- and time-dependent manners. The concentration required for 50 % inhibition (IC₅₀) was 1.15, 0.738, and 0.414 mg/ml, when cell were incubated with matrine for 24, 48, and 72 h, respectively. Matrine induced cell cycle arrest at G1 phase by downregulating cyclin D1 and upregulating p27 and p21. Matrine induced cell apoptosis by reducing the ratio of Bcl-2/Bax and increasing the activation of caspase-9 in a dose-dependent manner. Matrine displayed its anti-tumor activity by inactivating Akt, the upstream factor of the above proteins. Matrine significantly reduced the protein levels of pAkt, and increased the protein levels of other downstream factors, pBad and pGSK-3β. Specific inhibition of pAkt induced cell apoptosis, and synergized with matrine to inhibit the proliferation of LoVo cells; whereas activation of Akt neutralized the inhibitory effect of matrine on cell proliferation. The present study has demonstrated that matrine inhibits proliferation and induces apoptosis of human colon cancer LoVo cells by inactivating Akt pathway, indicating matrine may be a potential anti-cancer agent for colon cancer.     

DHA induces apoptosis by altering the expression and cellular location of GRP78 in colon cancer cell lines

n-3 polyunsaturated fatty acids exert growth-inhibitory and pro-apoptotic effects in colon cancer cells. We hypothesized that the anti-apoptotic glucose related protein of 78kDa (GRP78), originally described as a component of the unfolded protein response in endoplasmic reticulum (ER), could be a molecular target for docosahexaenoic acid (DHA) in these cells. GRP78 total and surface overexpression was previously associated with a poor prognosis in several cancers, whereas its down-regulation with decreased cancer growth in animal models. DHA treatment induced apoptosis in three colon cancer cell lines (HT-29, HCT116 and SW480), and inhibited their total and surface GRP78 expression. The cell ability to undergo DHA-induced apoptosis was inversely related to their level of GRP78 expression. The transfection of the low GRP78-expressing SW480 cells with GRP78-GFP cDNA significantly induced cell growth and inhibited the DHA-driven apoptosis, thus supporting the essential role of GRP78 in DHA pro-apoptotic effect. We suggest that pERK1/2 could be the first upstream target for DHA, and demonstrate that, downstream of GRP78, DHA may exert its proapoptotic role by augmenting the expression of the ER resident factors ERdj5 and inhibiting the phosphorylation of PKR-like ER kinase (PERK), known to be both physically associated with GRP78, and by activating caspase-4. Overall, the regulation of cellular GRP78 expression and location is suggested as a possible route through which DHA can exert pro-apoptotic and antitumoral effects in colon cancer cells.     

Curcumin inhibits the invasion of thyroid cancer cells via down-regulation of PI3K/Akt signaling pathway

The objectives of this study were to evaluate the in vitro anti-tumor (human thyroid cancer cell lines) potential of curcumin and to elucidate its molecular mechanisms. Here, we investigated the effects of curcumin on the cell viability, apoptosis, migration and invasion of human thyroid cancer cell lines FTC133. We also investigated the effects of curcumin on PI3K, p-Akt, MMP1/7, and COX-2 protein expressions using Western blot. Results showed that curcumin inhibited growth, cell migration and invasion in FTC133, and promoted its apoptosis. Western blot assay data demonstrated that curcumin inhibited phosphorylation of PI3K and Akt signaling pathways and subsequently attenuated MMP1/7 and COX-2 protein expressions in FTC133. In conclusion, curcumin suppresses FTC133 cell invasion and migration by inhibiting PI3K and Akt signaling pathways. Therefore, curcumin produces anti-metastatic activity in FTC133 cells.     

PI3K/Akt pathway involving into apoptosis and invasion in human colon cancer cells LoVo

In this study we determined the effects of Curcumin on human colon cancer cells line LoVo. We found that Curcumin significantly inhibited the proliferation, migration and invasion, and clone formation of LoVo cells in a dose-dependent manner. Curcumin also dose-dependently reduced the phosphorylation of proteins Akt and increased expression levels of the genes caspase-3, cytochrome-c, Bax mRNA in LoVo cells. In addition, Curcumin dose-dependently decreased gene Bcl-2 mRNA expression. Similar results were observed in LoVo cells treated with LY294002. These in vitro studies suggest that Curcumin may play its anti-cancer actions partly via suppressing PI3K/Akt signal pathway in LoVo cells.     

Alpha-lipoic acid induces apoptosis in hepatoma cells via the PTEN/Akt pathway

We report here that alpha-lipoic acid (alpha-LA), a naturally-occurring antioxidant, scavenges reactive oxygen species (ROS) followed by an increase in apoptosis of human hepatoma cells. Apoptosis induced by alpha-LA was dependent upon the activation of the caspase cascade and the mitochondrial death pathway. alpha-LA induced increases in caspase-9 and caspase-3 but had no significant effect on caspase-8 activity. Apoptosis induced by alpha-LA was found to be mediated through the tensin homologue deleted on chromosome 10 (PTEN)/Akt pathway. Prior to cell apoptosis, PTEN was activated and its downstream target Akt was inhibited. Our findings indicate that increasing ROS scavenging could be a therapeutic strategy to treat cancer.     

Apoptosis induction by wheat-flour sphingoid bases in DLD-1 human colon cancer cells

The apoptotic effects of plant sphingoid bases prepared from wheat-flour cerebroside on human colorectal cancer DLD-1 cells were examined. The viability of DLD-1 cells treated with such plant sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. Morphological changes such as condensed chromatin fragments were found, so those sphingoid bases reduced cell viability through causing apoptosis in these cells.     

Betanodavirus up-regulates chaperone GRP78 via ER stress: roles of GRP78 in viral replication and host mitochondria-mediated cell death

Whether viral pathogens that induce ER stress responses benefit the host or the virus remains controversial. In this study we show that betanodavirus induced ER stress responses up-regulate GRP78, which regulates the viral replication and host cellular mitochondrial-mediated cell death. Betanodavirus (redspotted grouper nervous necrosis virus, RGNNV) infection resulted in the following increased ER stress responses in fish GF-1 grouper fin cells: (1) IRE-1 and ATF-6 sensors at 48 h post-infection (p.i.) that up-regulated chaperone protein GRP78; (2) activation of caspase-12; and (3) PERK phosphorylation and down-regulation of Bcl-2. Analyses of GRP78 functions during viral replication using either loss-of-function or gain-of-function approaches showed that GRP78 over-expression also enhanced viral replication and induced cell death. Then, we found that zfGRP78 localization gradually increased in mitochondria after RGNNV infection by EGFP tagging approach. Furthermore, zfGRP78 can interact with viral RNA-dependent RNA polymerase (RdRp) by using immunofluorescent and immunoprecipitation assays. Finally, we found that blocking GRP78-mediated ER signals can reduce the viral death factors protein α and protein B2 expression and decrease the Bcl-2 down-regulation mediated mitochondria-dependent cell death, which also enhances host cellular viability. Taken together, our results suggest that RGNNV infection and expression can trigger ER stress responses, which up-regulate the chaperone GRP78 at early replication stage. Then, GRP78 can interact with RdRp that may enhance the viral replication for increasing viral death factors’ expressions at middle-late replication stage, which can enhance mitochondrial-mediated cell death pathway and viral spreading. These results may provide new insights into the mechanism of ER stress-mediated cell death in RNA viruses.     

Liposomal co-delivery-based quantitative evaluation of chemosensitivity enhancement in breast cancer stem cells by knockdown of GRP78/CLU

Resistance to chemotherapy is a key factor in the inefficacy of various forms of treatments for cancer. In the present study, chemo-resistant proteins, including glucose-regulated protein 78 (GRP78)/clusterin (CLU) targeted 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) liposomes, were developed as a delivery system for co-delivery of camptothecin (CPT) and GRP78 siRNA/CLU siRNA. Their drug/gene co-deliveries were quantitatively assessed in cancer stem cells (CSC) and MCF-7 cells. DOTAP-CPT/siRNA were prepared via electrostatic interaction on GRP78 siRNA or CLU siRNA. The size and ζ-potential of liposomes and lipoplexes were measured by dynamic light scattering techniques and electrophoretic light scattering spectrophotometry. The lipoplexes formation was tested by using gel electrophoresis. Immunofluorescence analysis showed that the expression level of CLU and GRP78 were significantly elevated in CSC compared to MCF-7 cells. Transfection and drug-delivery efficiency of DOTAP-CPT/siRNA were quantitatively compared with Lipofectamine 2000. Compared to free CPT, DOTAP-CPT-siCLU delivery in CSC and MCF-7 cells increased transfection efficiency and chemo-sensitivity by 4.1- and 5.9-fold, respectively. On the other hand, DOTAP-CPT-siGRP78 delivery increased transfection efficiency and chemo sensitivity by 4.4- and 6.2-fold in CSC and MCF-7 cells, respectively, compared to free CPT. It is significant that 3?±?1.2-fold increase in transfection efficiency was achieved by lipofectamine. Consequently, an increase in anti-cancer/gene silencing efficacy was quantitatively observed as an effect of DOTAP-CPT/siRNA treatment, which was relatively higher than lipofectamine treatment. Conclusively, our experimental data quantitatively demonstrate that using DOTAP-CPT-siRNA specifically targeting (CSCs) chemo-resistant protein in vitro offers substantial potential for synergistic anti-cancer therapy.     

Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway

A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serine-threonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo.