ROS-mediated apoptosis in colon cancer cells induced by sulfated polysaccharides from Gracilaria corticata

Sulfated polysaccharides (SPs) from red algae have attracted considerable interest due to their antioxidant and anticancer properties. Gracilaria corticata is a rich source of sulfated polysaccharides with notable antioxidant and anticancer potential. This study aimed to optimize the extraction process of SPs from G. corticata, determine their chemical and structural features, and evaluate their antioxidant and anticancer activities. The SPs were extracted from G. corticata using ultrasound-assisted extraction. The chemical composition, molecular weight (230 kDa), and structure of the extracted SPs were characterized using standard biochemical assays and nuclear magnetic resonance (NMR) spectroscopy. Antioxidant capacity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays. Cytotoxicity was assessed against HT-29 colon cancer cells and normal CCD-841 cells via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induction was further examined through Annexin V-FITC/PI staining and gene expression analysis of apoptosis-related markers. Ultrasound-assisted extraction was optimized at 12 min, 800 W power, and a solvent-to-biomass ratio of 30 mL/g, yielding 34.8% SPs. The SPs contained 84.4% carbohydrates, 8.7% sulfate, and 2.55% protein, with galactose identified as the predominant sugar. Molecular weight was determined to be 230 kDa. Antioxidant assays demonstrated dose-dependent activity, reaching 81.92% (DPPH) and 86.34% (ABTS) radical scavenging. SPs inhibited HT-29 cell viability with an IC₅₀ of 171 μg/mL and a selectivity index (SI) of 3.48. Apoptosis was induced via increased intracellular reactive oxygen species (ROS) and altered expression of key genes, including upregulation of Bax, P53, and Caspase 3, and downregulation of BCL2. SPs extracted from G. corticata, show strong antioxidant and selective anticancer activity through ROS-mediated apoptosis. These findings support their potential use as natural therapeutic agents in pharmaceutical development.

BackgroundMaslinic acid, a pentacyclic triterpene found in the protective wax-like coating of the leaves and fruit of Olea europaea L., is a promising agent for the prevention of colon cancer. We have shown elsewhere that maslinic acid inhibits cell proliferation to a significant extent and activates mitochondrial apoptosis in colon cancer cells. In our latest work we have investigated further this compound's apoptotic molecular mechanism.MethodsWe used HT29 adenocarcinoma cells. Changes genotoxicity were analyzed by single-cell gel electrophoresis (comet assay). The cell cycle was determined by flow cytometry. Finally, changes in protein expression were examined by western blotting. Student's t-test was used for statistical comparison.ResultsHT29 cells treated with maslinic acid showed significant increases in genotoxicity and cell-cycle arrest during the G0/G1 phase after 72 hours' treatment and an apoptotic sub-G0/G1 peak after 96 hours. Nevertheless, the molecular mechanism for this cytotoxic effect of maslinic acid has never been properly explored. We show here that the anti-tumoral activity of maslinic acid might proceed via p53-mediated apoptosis by acting upon the main signaling components that lead to an increase in p53 activity and the induction of the rest of the factors that participate in the apoptotic pathway. We found that in HT29 cells maslinic acid activated the expression of c-Jun NH2-terminal kinase (JNK), thus inducing p53. Treatment of tumor cells with maslinic acid also resulted in an increase in the expression of Bid and Bax, repression of Bcl-2, release of cytochrome-c and an increase in the expression of caspases -9, -3, and -7. Moreover, maslinic acid produced belated caspase-8 activity, thus amplifying the initial mitochondrial apoptotic signaling.ConclusionAll these results suggest that maslinic acid induces apoptosis in human HT29 colon-cancer cells through the JNK-Bid-mediated mitochondrial apoptotic pathway via the activation of p53. Thus we propose a plausible sequential molecular mechanism for the expression of the different proteins responsible for the intrinsic mitochondrial apoptotic pathway. Further studies with other cell lines will be needed to confirm the general nature of these findings.

Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C(2)-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C(2)-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C(2)-ceramide inhibited growth and caused death of colon cancer cells in time- and concentration-dependent manners, whereas C(2)-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C(2)-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C(2)-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C(2)-ceramide increased the number of cells in the A(0) peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G(2)/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis in colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis.

BackgroundIn an effort to achieve better cancer therapies, we elucidated the combination cancer therapy of STI571 (an inhibitor of Bcr-Abl and clinically used for chronic myelogenous leukemia) and TNF-related apoptosis-inducing ligand (TRAIL, a developing antitumor agent) in leukemia, colon, and prostate cancer cells.MethodsColon cancer (HCT116, SW480), prostate cancer (PC3, LNCaP) and leukemia (K562) cells were treated with STI571 and TRAIL. Cell viability was determined by MTT assay and sub-G1 appearance. Protein expression and kinase phosphorylation were determined by Western blotting. c-Abl and p73 activities were inhibited by target-specific small interfering (si)RNA. In vitro kinase assay of c-Abl was conducted using CRK as a substrate.ResultsWe found that STI571 exerts opposite effects on the antitumor activity of TRAIL. It enhanced cytotoxicity in TRAIL-treated K562 leukemia cells and reduced TRAIL-induced apoptosis in HCT116 and SW480 colon cancer cells, while having no effect on PC3 and LNCaP cells. In colon and prostate cancer cells, TRAIL caused c-Abl cleavage to the active form via a caspase pathway. Interestingly, JNK and p38 MAPK inhibitors effectively blocked TRAIL-induced toxicity in the colon, but not in prostate cancer cells. Next, we found that STI571 could attenuate TRAIL-induced c-Abl, JNK and p38 activation in HCT116 cells. In addition, siRNA targeting knockdown of c-Abl and p73 also reduced TRAIL-induced cytotoxicity, rendering HCT116 cells less responsive to stress kinase activation, and masking the cytoprotective effect of STI571.ConclusionsAll together we demonstrate a novel mediator role of p73 in activating the stress kinases p38 and JNK in the classical apoptotic pathway of TRAIL. TRAIL via caspase-dependent action can sequentially activate c-Abl, p73, and stress kinases, which contribute to apoptosis in colon cancer cells. Through the inhibition of c-Abl-mediated apoptotic p73 signaling, STI571 reduces the antitumor activity of TRAIL in colon cancer cells. Our results raise additional concerns when developing combination cancer therapy with TRAIL and STI571 in the future.

SP1-induced circ_0017552 modulates colon cancer cell proliferation and apoptosis via up-regulation of NET1

Autophagy is a catabolic process by which a cell degrades its intracellular materials to replenish itself. Induction of autophagy under various cellular stress stimuli can lead to either cell survival or cell death via apoptotic and/or autophagic (nonapoptotic) pathways. The NSAID sulindac sulfide induces apoptosis in colon cancer cells. Here, we show that inhibition of autophagy under serum-deprived conditions resulted in significant reductions of sulindac sulfide-induced apoptosis in HT-29 colon cancer cells. In contrast, inhibition of autophagy under conditions where serum is available significantly increased sulindac sulfide-induced apoptosis in HT-29 cells. We previously showed that the apoptosis inhibitor, survivin, plays a role in regulating NSAID-induced apoptosis and autophagic cell death. Here, we show that survivin protein half-life is increased in the presence of autophagy inhibitors under serum-deprived conditions, but not under conditions when serum is available. Thus, the increased levels of survivin may be a factor contributing to inhibition of sulindac sulfide-induced apoptosis under serum-deprived conditions. These results suggest that whether a cell lives or dies due to autophagy induction depends on the balance of factors that regulate both autophagic and apoptotic processes.

Epidemiological studies have demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of colon cancer. In addition, NSAIDs reduce the number and size of polyps in patients with familial adenomatous polyposis. The mechanisms of the anti-neoplastic effect of NSAIDs are still far from complete understanding, but one possible mechanism is the induction of apoptosis. Several lines of evidence suggest that NSAIDs-induced apoptosis in colon cancer cells are mediated through the cyclooxygenase (COX)-independent pathway. In this study we explored the mechanism of NSAIDs-induced apoptosis in the colon cancer cell line, HT-29. We confirmed that NSAIDs induce apoptosis in HT-29 cells irrespective of their COX-selectivity. Indomethacin enhanced the expression of p21waf-1 in HT-29 cells. However the expression of apoptosis-related genes such as Fas, bcl-2 and bax was not affected by indomethacin. Intra- and extra-cellular calcium chelators, protein tyrosine kinase (PTK) inhibitor, protein kinase A (PKA) inhibitor and protein kinase C (PKC) inhibitors did not influence indomethacin-induced apoptosis in HT-29 cells. We concluded that NSAIDs-induced apoptosis in colon cancer cells may be independent from signals transducted through [Ca++]i, PTK, PKA, PKC or the expression of apoptosis-related genes. In contrast, our results demonstrating the induction of p21waf-1 transcription by NSAIDs suggest the possible association of NSAIDs-induced apoptosis and cell-cycle control in colon cancer cells.

This study used both in vitro and in vivo models to evaluate the efficacy of atractylodin as an anticancer treatment for colorectal cancer. The cytotoxicity of atractylodin on colon cancer cells was assessed using the MTT assay, and atractylodin-induced apoptosis was determined using flow cytometry. The expression of cleaved caspase 3 and other apoptotic proteins was examined using Western blotting to determine the mechanism underlying atractylodin's anticancer activity. In addition, the role of PI3K/Akt/mTOR/p70S6K signalling in atractylodin-induced apoptosis in colon cancer cells was analyzed. The study found that atractylodin caused dose-dependent ROS-mediated apoptosis and DNA damage in colon cancer cells and activated caspase 3. Furthermore, atractylodin inhibited the PI3K/Akt/mTOR/p70S6K signalling pathway by targeting PI3Kγ in colon cancer cells. Molecular docking analysis indicated that atractylodin binds to the Akt binding pocket of PI3Kγ. The study also evaluated the antitumour effects of atractylodin on a colon cancer tumour xenograft model and found that it significantly reduced tumour growth and volume by inducing apoptosis. These results suggest that atractylodin has potential as a candidate for the treatment of colorectal cancer, although further research is necessary. Graphical abstract Atractylodin induces apoptosis in colon cancer cells.

Colorectal cancer has become more common in many regions of the world. Recently, we showed that esculetin, a natural coumarin, induces apoptosis in HT-29 colon cancer cells via the reactive oxygen species-mediated mitochondrial pathway. The present study examined whether esculetin induces apoptosis in HT-29 colon cancer cells by inducing endoplasmic reticulum (ER) stress. We found that esculetin induced characteristic signs of ER stress, confirmed by ER staining, mitochondrial calcium overload and expression of ER stress-related proteins (i.e. glucose regulated protein 78, phosphorylated ribonucleic acid-dependent protein kinase-like ER kinase, phosphorylated inositol requiring enzyme 1, phosphorylated eukaryotic initiation factor-2α, spliced X-box binding protein 1 and cleaved activating transcription factor 6). Esculetin also induced the expression of the CCAAT/enhancer-binding protein-homologous protein (CHOP) and pro-apoptotic factors caspase-12. Moreover, transfection of colon cancer cells with a small interfering ribonucleic acid targeting CHOP attenuated esculetin-induced apoptosis. Taken together, these results suggest that the ER stress response plays an important role in esculetin-induced apoptosis in human colon cancer cells.

Objective To investigate the apoptosis of colonic cancer cells co-cultured with T lymphocytes induced by dendritic cells modified by proliferating cell nuclear antigen (PCNA)-Epitope and interleukin-18 (IL-18) genes.Methods The experiment was divided into four groups,including DCs group,empty vector group,PCNA-Epitope transfection group,and PCNA-Epitope plus IL-18 gene group. Everygroup was operated according to DCs vs.T ratio (1∶ 10).The empty vector plasmid (pIRES-EGFP),PCNA-Epitope plasmid [ plRES-EGFP-PCNA(6) ] and plasmid loaded with PCNA-Epitope and IL-18 gene [ pIRES-EGFP-PCNA(6)/IL-18 ] were transfected into peripheral blood-derived dendritic cells by non-lipid polymer-based reagent,then co-cultured with T lymphocytes,respectively.The supernatants of immunocytes were cultured with colonic cancer cells according to the ratio of T lymphocytes vs.colonic cancer cells (5∶ 1,10∶1,20∶1,40∶1 ).The apoptosis of colonic cancer cells was examined by using flow cytometry.Results The apoptosis of colonic cancer cells could be induced in every group. The apoptosis in the pIRES-EGFP-PCNA(6)/IL-18 group was significantly higher than in DCs group,empty vector group,and PCNA-Epitope transfection group.The optimal ratio of T lymphocytes vs.colonic cancer cells was 40∶ 1.Conclusion The supernatants of T lymphocytes induced by DCs that were treated with the PCNA-Epitope and IL-18 gene can induce apoptosis of more colonic cancer cells. Key words: Colonic carcinoma; Dendritic cells; Proliferating cell nuclear antigen; Interleukin-18; Apoptosis

Camptothecin (CPT) is an anti-cancer drug that effectively treats various cancers, including colon cancer. However, poor solubility and other drawbacks have restricted its chemotherapeutic potential. To overcome these restrictions, CPT was encapsulated in CEF (cyclodextrin-EDTA-FE3O4), a composite nanoparticle of magnetic iron oxide (Fe3O4), and β-cyclodextrin was cross-linked with ethylenediaminetetraacetic acid (EDTA). This formulation improved CPT’s solubility and bioavailability for cancer cells. The use of magnetically responsive anti-cancer formulation is highly advantageous in cancer chemotherapy. The chemical characterisation of CPT-CEF was studied here. The ability of this nano-compound to induce apoptosis in HT29 colon cancer cells and A549 lung cancer cells was evaluated. The dose-dependent cytotoxicity of CPT-CEF was shown using MTT. Propidium iodide and Annexin V staining, mitochondrial membrane depolarisation (JC-1 dye), and caspase-3 activity were assayed to detect apoptosis in CPT-CEF-treated cancer cells. Cell cycle analysis also showed G1 phase arrest, which indicated possible synergistic effects of the nano-carrier. These study results show that CPT-CEF causes a dose-dependent cell viability reduction in HT29 and A549 cells and induces apoptosis in colon cancer cells via caspase-3 activation. These data strongly suggest that CPT could be used as a major nanocarrier for CPT to effectively treat colon cancer.

In this study, we aimed to observe the effect of polyamidoamine (PAMAM) liposomes on the apoptosis of human colon cancer cells induced by survivin antisense oligonucleotides (ASODNs). PAMAM liposomes and PAMAM were mixed with survivin ASODNs to obtain antisense gene transfection complexes. In addition, the zeta potentials and encapsulation rates of the complexes were measured. The two gene-containing complexes were transfected into HT-29 colon cancer cells to observe changes in cell morphology, detect the inhibitory effect on tumor cells and changes in apoptosis, and observe changes in the cytoskeleton microfilament system using laser confocal microscopy. Caspase-3 activity in the cells was determined using a kinase activity assay, and p38 mitogen-activated protein kinase (p38 MAPK) activity in the cells was measured using immunoprecipitation analysis. The results showed that the zeta potential of the PAMAM liposome-survivin-ASODN complex was higher than that of the PAMAM-survivin-ASODN complex (P < 0.05). There was no significant difference in the gene encapsulation rates between the two complexes (P > 0.05). PAMAM liposomes may efficiently deliver survivin ASODNs to human colon cancer cells, reduce the expression of survivin protein and at the same time induce G2/M phase arrest in cells, and activate caspase-3 by activating p38 MAPK. Cleavage of caspase-3 destroys the structure of the intracellular skeletal microfilament system, finally resulting in apoptosis of colon cancer cells.

Butyrate, a fermentation product of fiber in the colon, acts as a histone deacetylase inhibitor (HDACi) and induces apoptosis in colon cancer (CC) cells in vitro. We have reported that the apoptotic effects of butyrate are dependent upon the hyperactivation of the Wnt/beta-catenin pathway. However, prolonged exposure of CC cells to increasing concentrations of butyrate results in the acquisition of resistance to the Wnt/beta-catenin- and apoptosis-inducing effects of this agent, as well as cross-resistance to structurally different HDACis. Here we report that one mechanism whereby HDACi resistance arises is through the increase of beta-catenin-independent (noncanonical) Wnt signaling. Compared to HDACi-sensitive HCT-116 CC cells, HDACi-resistant HCT-R cells exhibit higher levels of AKT/PKB cell survival signaling, which is in part induced by WNT5A and its receptor ROR2. The induction of AKT signaling by HDACis is also detected in other CC cell lines, albeit to a lesser extent than in the drug-resistant HCT-R cells. The observations suggested that the apoptotic effect of butyrate and other HDACis in CC cells can be augmented by inhibitors of pAKT. In agreement with the hypothesis, the combination of MK2206, a pAKT inhibitor, and a HDACi (butyrate or LBH589) induced higher apoptosis in CC cells compared to each agent alone. The exposure to both agents also re-sensitized the HCT-R cells to apoptosis. Finally, the concept of simultaneously inducing canonical Wnt activity and suppressing AKT signaling was translated into a combination of diet-derived agents. Diet-derived pAKT inhibitors (caffeic acid phethyl ester, sulforaphane, dilallyl trisulfide) suppressed the butyrate-induced levels of pAKT, and increased the apoptotic effects of butyrate in both drug-sensitive and drug-resistant CC cells.Our findings can be translated into (a) CC therapy employing combinations of synthetic HDACis and inhibitors of pAKT, as well as (b) CC prevention based upon diets that result in sufficient amounts of butyrate and pAKT inhibitors.

The aim of this study was to investigate the effect of cullin 4A (CUL4A) on the proliferation and apoptosis of colon cancer (CC) cells, and to elucidate its regulatory relationship with the Hippo pathway. Paired CC tissues and adjacent normal tissues were obtained from patients. CC cells were isolated and cultured in vitro. CUL4A was interfered by small interfering ribonucleic acid (siRNA) (siR-CUL4A group) or overexpressed by overexpression vector (CUL4A-Vector group), with negative control (NC)-CUL4A or CUL4A -NC as the control group. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression level of CUL4A in CC tissues and cells. The proliferative ability of cells was detected by cell counting kit-8 (CCK-8) assay. Flow cytometry was applied to measure the apoptosis of cells in each group. Western blotting (WB) was conducted to determine the protein expression of CUL4A. In addition, the proliferative ability was examined in vivo through subcutaneous injection of cells into nude mice. QRT-PCR showed that CUL4A was highly expressed in 66.67% of CC samples (p<0.01). In vivo and in vitro proliferative ability was significantly reduced in siR-CUL4A group (p<0.01), whereas the apoptosis rate was promoted (p<0.01). However, in vivo and in vitro proliferative ability increased significantly in CUL4A-Vector group (p<0.01), while the apoptosis rate was reduced (p<0.01). The protein expressions of MST1, LATS1 and p-YAP were significantly up-regulated in siR-CUL4A group (p<0.01), while they were remarkably down-regulated in CUL4A-Vector group (p<0.05, p<0.01). CUL4A is highly expressed in CC and promotes the proliferation and inhibits the apoptosis of CC cells by regulating the Hippo pathway.

Apoptosis plays a central role in tumor development and it has been hypothesized that lack/failure of apoptosis leads to the development of tumors, including colon tumors. Thus, induction of apoptosis in tumor cells is an effective approach to the regulation of tumor growth. It has been shown by us and other investigators that various chemopreventive agents induce apoptosis and inhibit tumor growth. Identification of agents or combinations of agents that induce tumor cell apoptosis guides the development of novel agents for colon cancer treatment. Experiments were designed to assess the effectiveness of lovastatin, a 3-hydroxy-3-methyl glutaryl-CoA reductase inhibitor, and celecoxib a cyclooxygenase-2 inhibitor, individually or in combination on the induction of apoptosis in human HT-29 colon cancer cells. In addition, we studied the modulatory effect of lovastatin and celecoxib on lamin B levels, caspase-3 activity and expression in relationship to apoptosis in colon cancer cell lines. HT-29 cells exposed to various subtoxic levels of lovastatin or celecoxib or a combination of both were analyzed for apoptosis (by DAPI method), caspase-3 expression (immunoblot analysis) and caspase-3 activity (fluorimetric method). We found that: i) pretreatment with lovastatin (5-30 microM) induces apoptosis in HT-29 cells significantly only at high concentrations (> or = 20 microM) but not at low dose levels; ii) similarly, pretreatment with celecoxib produced apoptosis in colon cancer cells at high concentrations only (> or = 75 microM); iii) caspase-3 protein expression was moderately altered by the treatment with lovastatin or celecoxib at lower concentrations; however, a significant increase (1.6 to 4-fold) in caspase-3 expression and activity was found in HT-29 cells exposed with 20-25 microM lovastatin and/or 5-125 microM celecoxib and iv) importantly, in tumor cells exposed to low doses of (5 or 10 microM) lovastatin, combined with 25-75 microM of celecoxib, apoptosis induction rose 2.5 to 10-fold, caspase-3 expression was 2.3 to 8-fold higher, and enzyme activities were 1.5 to 5.5-fold elevated. This effect was highly synergistic and dose-dependent. Lamin B levels were significantly increased in a dose-dependent manner in cells treated with lovastatin but no such effect was observed with celecoxib. These results indicate that agents with different modes of action when applied in combinations will induce apoptosis synergistically by enhancing caspase-3 activities. These findings further support the hypothesis that HMGCo-R and COX-2 activities play important roles in apoptosis and regulation of apoptosis by selective agents such as lovastatin and celecoxib would provide effective strategies for the prevention of colon cancer.