Prodigiosin-induced apoptosis in human colon cancer cells

Prodigiosin is a red pigment produced by various bacteria including Serratia marcescens. Colorectal cancer is one of the most frequent malignancies and one of the most frequent causes of cancer death in the Western world. Its treatment is far from satisfactory and the challenge to oncologists is to find novel chemical entities with less toxicity and greater effectiveness than those used in current chemotherapy. Here we characterize the apoptotic action of prodigiosin in colon cancer cells. DLD-1 and SW-620 human colon adenocarcinoma cells, NRK and Swiss-3T3 nonmalignant cells were assayed by the MTT assay, fragmentation pattern of DNA, Hoechst 33342 staining and study of PARP cleavage by Western blot, in order to characterize the prodigiosin-induced apoptosis. Prodigiosin was purified and its structure was confirmed. Metastatic SW-620 cells were more sensitive to prodigiosin (IC50: 275 nM) than DLD-1. We did not observe a significant decrease in the viability of NRK cells. We confirmed that prodigiosin induces apoptosis in both cancer cell lines by the characteristic DNA laddering pattern and condensed nuclei or apoptotic bodies identified by fluorescence microscopy. These results indicate that prodigiosin induces apoptosis in colon cancer cells.     

Alpha-tocopheryl succinate sensitizes human colon cancer cells to exisulind-induced apoptosis

Sulindac sulfone (also known as exisulind) and its chemical derivatives are promising anticancer agents capable of inducing apoptosis in a variety of malignant cell types with minimal toxicity to normal cells. Here, we tested the ability of alpha-tocopheryl succinate (TOS), another promising anticancer agent, to sensitize colon cancer cells to exisulind-induced apoptosis. We found that sub-apoptotic doses of TOS greatly enhanced exisulind-induced growth suppression and apoptosis in the HCT116, LoVo and SNU-C4 human colon cancer cell lines. Our results revealed that this was accounted for primarily by an augmented cleavage of poly(ADP-ribose) polymerase (PARP) and enhanced activation of caspase-8, -9 and -3. Pretreatment with z-VAD-FMK (a pan-caspase inhibitor), z-IETD-FMK (a caspase-8 inhibitor) or z-LEHD-FMK (a caspase-9 inhibitor) blocked TOS and exisulind cotreatment-induced PARP cleavage and apoptosis. Furthermore, TOS/exisulind cotreatment induced JNK phosphorylation, while pretreatment with SP600151 (a JNK inhibitor) partially blocked cotreatment-induced caspase-dependent PARP cleavage and apoptosis. Taken together, these findings indicate that TOS sensitizes human colon cancer cells to exisulind-induced apoptosis. Apoptotic synergy induced by exisulind plus TOS seems likely to be mediated through a mechanism involving activation of caspases and JNK. S.-J. Lim, Y.-J. Lee both authors are contributed equally to this study.     

TNF-alpha-mediated apoptosis in vascular smooth muscle cells requires p73

Atherosclerosis, now considered an inflammatory process, is the leading cause of death in the Western world and is manifested by a variety of diseases in multiple organ systems. Because of its prevalence and associated morbidity, novel therapies directed at arresting this progressive process are urgently needed. The inflammatory mediator TNF-, which is known to contribute to apoptosis in vascular smooth muscle cells, has been shown to be intimately involved in the atherosclerotic process, being present at elevated levels in human atheroma as well as possibly being responsible for plaque rupture, a clinically devastating event. In light of our earlier finding that p73 is a proapoptotic protein in vascular smooth muscle cells, which are involved in plaque progression as well as rupture, we asked whether TNF- mediates apoptosis in these cells through p73. We now show that p73 is present in spindle-shaped cells within human atheroma, and p73, an isoform that is pivotal in both apoptosis and growth suppression, is induced in vascular smooth muscle cells in vitro by serum but not by PDGF-BB. In addition, TNF-, when added to these cells in the presence of serum-containing media, increases p73 expression and causes apoptosis in both rat and human vascular smooth muscle cells. Inhibition of p73 activity with a dominant inhibitory NH₂-terminally deleted p73 plasmid results in markedly decreased TNF--induced apoptosis. Thus p73 is likely a mediator of the apoptotic effect of TNF- in the vasculature, such that future targeting of the p73 isoforms may ultimately prove useful in novel atherosclerosis therapies. atherosclerosis; inflammation; plaque     

ROS mediate selenite-induced apoptosis in colon cancer cells

Selenite-induced oxidative stress and its relationship to mitochondrial apoptosis was studied in human adenocarcinoma HT-29 cells. It is shown that selenite induces caspase-dependent apoptosis, which is mediated by mitochondria via released cytochrome c, apoptosis-inducing factor (AIF) and Smac/Diablo. Selenite activates stress kinases p38 and JNK while suppressing reduced glutathione (GSH) and thioredoxin reductase (TrxR) levels, transiently inducing heme oxygenase (HO-1) system as well as reducing Akt expression. Pre-treatment of cells with selected antioxidants and stress kinase inhibitors significantly prevented selenite-induced cell death, thereby implicating oxidative stress as a direct (Bax) as well as indirect (via kinases) cause of HT-29 cells demise. These results thus demonstrate for the first time active proapoptotic and anti-survival effects of selenite in colon cancer cells.     

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.     

Indomethacin and juglone inhibit inflammatory molecules to induce apoptosis in colon cancer cells

Colorectal cancer (CRC) is the third most common fatal cancer. Indomethacin, a nonsteroidal anti‐inflammatory drug, is known to reduce the occurrence of CRC. This study evaluated the potential anticolon cancer effects of juglone (5‐hydroxy‐1,4‐naphthoquinone) in combination with indomethacin. Human colon adenocarcinoma cells (HT29) were subjected to treatment with indomethacin, juglone, and a combination of both. Morphological analysis, cell cycle regulation, and dual staining using acridine orange and ethidium bromide in control and treated cells revealed the apoptotic potential of these compounds. Bcl2 and inflammatory molecules (tumor necrosis factor‐α, nuclear factor kappa B, and Cox‐2) were found to be decreased with a concomitant increase in the expression of proapoptotic molecules (Bad, Bax, cytochrome c, and PUMA) as a result of the molecular regulation of Wnt, Notch, and peroxisome proliferator‐activated receptor‐γ signaling. Treatment with juglone was not as effective as with indomethacin; however, a combination of both was shown to be more effective, suggesting that juglone may be considered for therapeutic intervention of colon cancer.     

A new EGFR inhibitor induces apoptosis in colon cancer cells

The use of agents targeting EGFR represents a new frontier in colon cancer therapy. Among these, mAbs and EGFR tyrosine kinase inhibitors seemed to be the most promising. However they have demonstrated scarce utility in therapy, the former being effective only at toxic doses, the latter resulting inefficient in colon cancer. This paper presents studies on a new EGFR inhibitor, FR18, a molecule containing the same naphthoquinone core as shikonin, an agent with great anti-tumor potential. In HT29, a human colon carcinoma cell line, flow cytometry, immunoprecipitation, and Western blot analysis, confocal spectral microscopy have demonstrated that FR18 is active at concentrations as low as 10 nM, inhibits EGF binding to EGFR while leaving unperturbed the receptor kinase activity. At concentration ranging from 30 nM to 5 microM, it activates apoptosis. FR18 seems therefore to have possible therapeutic applications in colon cancer.     

Dictyopteris undulata extract induces apoptosis in human colon cancer cells

The present study investigated the cytotoxic and apoptotic effects of an ethanol extract derived from the marine brown alga Dictyopteris undulata against human colon adenocarcinoma cells. The Dictyopteris undulata extract (DUE) showed cytotoxic activity against SW480 cells in a dose-dependent manner, with 50% inhibition of cell viability at a concentration of 40 μg/mL. DUE also induced programmed cell death in SW480 cells, as evidenced by apoptotic body formation, DNA fragmentation, an increase in the population of apoptotic sub-G1 phase cells, and mitochondrial membrane depolarization. Moreover, DUE significantly modulated the expression of apoptosisassociated proteins, resulting in a decrease in B cell lymphoma-2 expression and an increase in Bcl-2-associated X protein expression, as well as the activation of caspase-9 and caspase-3. Furthermore, DUE showed apoptotic cell death in two other colon cancer cell lines, SNU407 and HT29. These observations suggest that DUE may prove useful as a therapeutic agent for the attenuation of colon cancer.     

Isorhamnetin in Tsoong blocks Hsp70 expression to promote apoptosis of colon cancer cells

The roots of Codonopis bulleynana Forest ex diels (cbFed), locally known as Tsoong, have been used as a tonic food. Tsoong has wide range of pharmacological effects, including anticancer efficacy. In the present study, the anticancer activity of Tsoong and its potential molecular mechanisms were investigated. Isorhamnetin, a flavonol aglycone, is important compound and metabolite in Tsoong. It can promote apoptosis of colon cancer cells through up-regulating apoptosis-related genes (Apaf1, Casp3 and Casp9) because it blocks Hsp70 genes (Hspa1a, Hspa1b and Hspa8). These were verified by in vitro and in vivo experiments. In vitro, cell counting kit-8 (CCK-8) assays and flow cytometry in HCT116 and SW480 colon cancer cell were used to assess the anti-proliferation and apoptosis-promoting activities of Tsoong. In vivo, the antitumor effect of Tsoong was assessed in colon cancer-bearing nude mice as a xenograft model. These results show that Isorhamnetin is very critical in Tsoong because Tsoong can down-regulate Hsp70 genes and promote apoptosis of colon cancer cells by inhibiting Hsp70 largely due to the efficacy of Isorhamnetin. Our results may ultimately help in the development of diagnostic and therapeutic strategies to control this devastating disease.     

PUMA Dissociates Bax and Bcl-X(L) to induce apoptosis in colon cancer cells

PUMA is a BH3-only Bcl-2 family protein that plays an essential role in DNA damage-induced apoptosis. PUMA interacts with anti-apoptotic Bcl-2 and Bcl-X(L) and is dependent on Bax to induce apoptosis. In this study, we investigated how the interactions of PUMA with the antiapoptotic proteins coordinate with Bax to initiate apoptosis in HCT116 colon cancer cells. We found that Bcl-X(L) was most effective among several antiapoptotic proteins in suppressing PUMA-induced apoptosis and PUMA-dependent apoptosis induced by the DNA-damaging agent adriamycin. Mutant Bcl-X(L) that cannot interact with Bax was unable to protect cells from PUMA-mediated apoptosis. Knockdown of Bcl-X(L) by RNA interference significantly enhanced PUMA-mediated apoptosis in HCT116 cells but not in PUMA-knockout cells. Furthermore, Bax was found to be dissociated preferentially from Bcl-X(L) in HCT116 cells but not in the PUMA-knockout cells, in response to PUMA induction and adriamycin treatment. PUMA inhibited the association of Bax and Bcl-X(L) in vitro by directly binding to Bcl-X(L) through its BH3 domain. Finally, we found that wild-type Bax, but not mutant Bax deficient in either multimerization or mitochondrial localization, was able to restore PUMA-induced apoptosis in the BAX-knockout cells. Together, these results indicate that PUMA initiates apoptosis in part by dissociating Bax and Bcl-X(L), thereby promoting Bax multimerization and mitochondrial translocation.     

TNF-alpha-mediated cardiomyocyte apoptosis involves caspase-12 and calpain

Following ischemia-reperfusion, there is a sustained increase of TNF-alpha both locally in the heart as well as in circulating levels in blood. While TNF-alpha has been implicated in cardiomyocyte apoptosis which occurs in several cardiomyopathies, the molecular pathways by which TNF-alpha induces apoptosis in these cells are not fully elucidated. We investigated the role of the two families of cysteine proteases, caspases and calpains, which are known to participate in apoptotic cell death. The effect of the highly specific calpain inhibitor, Z-LLY-fmk, and the caspase pathways involved in TNF-alpha-mediated apoptosis of the HL-1 cardiomyocyte cell line were examined. Activation of the downstream caspase-3, and the cleavage of poly ADP-ribose polymerase (PARP) were observed in a time-dependent manner upon treatment with TNF-alpha. Caspase-12, but not caspase-9, was activated in response to TNF-stimulation, indicating that an endoplasmic reticulum (ER)/calcium-dependent pathway may be involved. In HL-1 cardiomyocytes, TNF-alpha-induced apoptosis appears to be mediated by calpain as apoptotic changes were abrogated in the presence of the highly specific calpain inhibitor, Z-LLY-fmk. In conclusion, our results suggest that TNF-alpha-mediated apoptosis in HL-1 cardiomyocytes follows the caspase-12 apoptotic pathway that involves calpain.     

Lobetyolin induces apoptosis of colon cancer cells by inhibiting glutamine metabolism

The purpose of the present study was to evaluate the anti-cancer property of Lobetyolin on colorectal cancer and explore its potential mechanism. Lobetyolin was incubated with HCT-116 cells in the absence or presence of ASCT2 inhibitor Benser or p53 inhibitor Pifithrin-α. The levels of glutamine, glutamic acid, α-ketoglutarate, ATP and GSH were determined to measure the glutamine metabolism. Annexin V-FITC/PI staining and TUNEL assay were applied to estimate the apoptotic condition. The levels of ASCT2 were examined by RT-qPCR, Western blot and immunofluorescence staining. The expressions of cleaved-caspase-3, caspase-3, cleaved-caspase-7, caspase-7, cleaved-PARP, PARP, p53, p21, bax and survivin were detected using Western blot analysis. As a result, the treatment with Lobetyolin effectively induced apoptosis and glutamine metabolism in HCT-116 cells through ASCT2 signalling. The inhibition of ASCT2 reduced the glutamine-related biomarkers and augmented the apoptotic process. We further found that the effect of Lobetyolin on HCT-116 was related to the expressions of p21 and bax, and transportation of p53 to nucleus. The inhibition of p53 by Pifithrin-α promoted the inhibitory effect of Lobetyolin on ASCT2-mediated apoptosis. Lobetyolin also exerted anti-cancer property in nude mice. In conclusion, the present work suggested that Lobetyolin could induce the apoptosis via the inhibition of ASCT2-mediated glutamine metabolism, which was possibly governed by p53.     

Ethanol acts as a potent agent sensitizing colon cancer cells to the TRAIL-induced apoptosis

Identification of mechanisms of modulation of the TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis is important for its potential use in anticancer therapy. Ethanol can induce cell death in vitro and in vivo by different signalling pathways. Its effect in combination with death ligands is unknown. We investigated how ethanol modulates the effects of TRAIL in colon cancer cells. After combined TRAIL and ethanol treatment, a potentiation of caspase-8, -9, -3 activation, a proapoptotic Bid protein cleavage, a decrease of mitochondrial membrane potential, a complete poly(ADP)ribose polymerase cleavage, and disappearance of antiapoptotic Mcl-1 protein were demonstrated. Ethanol acts as a potent agent sensitizing colon cancer cells to TRAIL-induced apoptosis.     

Resveratrol induces mitochondrial respiration and apoptosis in SW620 colon cancer cells

BackgroundThe polyphenol resveratrol (RSV) is found in the skin of red grapes and has been reported to exhibit anticancer properties. The antitumor effects of RSV in the gastrointestinal tract have gained considerable interest due to the high exposure of this tissue to this dietary compound. One of the hallmarks of cancer cells is their particular metabolism mainly relying on glycolysis for ATP production rather than mitochondrial oxidative phosphorylation. Although RSV has been described to act as a calorie-restriction mimetic, modulating energy metabolism in normal tissues, little efforts have been done to study the effects of this polyphenol in the metabolism of cancer cells. Taking this into account, the aim of this study was to explore metabolic effects of this polyphenol in colon cancer.MethodsOxygen consumption, ATP levels, Western blotting and other molecular biology techniques were carried out to characterize the metabolic signature of RSV in SW620 colon cancer cells.ResultsParadoxically, the cytotoxic effects of RSV were associated with an increase in oxygen consumption supported by mitochondrial biogenesis and increased fatty acid oxidation. This partial reversion of the Warburg effect was followed by hyperpolarization of mitochondrial membrane and ROS production, leading to an increased apoptosis.ConclusionsOur results propose that the anticancer mechanisms of RSV could reside in targeting cancer cell metabolism, promoting mitochondrial electron transport chain overload and, ultimately, increasing ROS production.General significanceThese results shed new light into the anticancer mechanism of RSV supporting the ability of this compound in potentiating the effects of chemotherapy.     

beta-lapachone induces cell cycle arrest and apoptosis in human colon cancer cells

BACKGROUND: Human colon cancers have a high frequency of p53 mutations, and cancer cells expressing mutant p53 tend to be resistant to current chemo- and radiation therapy. It is thus important to find therapeutic agents that can inhibit colon cancer cells with altered p53 status. beta-Lapachone, a novel topoisomerase inhibitor, has been shown to induce cell death in human promyelocytic leukemia and prostate cancer cells through a p53-independent pathway. Here we examined the effects of beta-lapachone on human colon cancer cells. MATERIALS AND METHODS: Several human colon cancer cell lines, SW480, SW620, and DLD1, with mutant or defective p53, were used. The antiproliferative effects of beta-lapachone were assessed by colony formation assays, cell cycle analysis, and apoptosis analysis, including annexin V staining and DNA laddering analysis. The effects on cell cycle and apoptosis regulatory proteins were examined by immunoblotting. RESULTS: All three cell lines, SW480, SW620, and DLD1, were sensitive to beta-lapachone, with an IC(50) of 2 to 3 microM in colony formation assays, a finding similar to that previously reported for prostate cancer cells. However, these cells were arrested in different stages of S phase. At 24 hr post-treatment, beta-lapachone induced S-, late S/G2-, and early S-phase arrest in SW480, SW620, and DLD1 cells, respectively. The cell cycle alterations induced by beta-lapachone were congruous with changes in cell cycle regulatory proteins such as cyclin A, cyclin B1, cdc2, and cyclin D1. Moreover, beta-lapachone induced apoptosis, as demonstrated by annexin V staining, flow cytometric analysis of DNA content, and DNA laddering analysis. Furthermore, down-regulation of mutant p53 and induction of p27 in SW480 cells, and induction of pro-apoptotic protein Bax in DLD1 cells may be pertinent to the anti-proliferative and apoptotic effects of beta-lapachone on these cells. CONCLUSIONS: beta-Lapachone induced cell cycle arrest and apoptosis in human colon cancer cells through a p53-independent pathway. For human colon cancers, which often contain p53 mutations, beta-lapachone may prove to be a promising anticancer agent that can target cancer cells, especially those with mutant p53.     

Effects of silk sericin on the proliferation and apoptosis of colon cancer cells

Sericin is a silk protein woven from silkworm cocoons (Bombyx mori). In animal model, sericin has been reported to have anti-tumoral action against colon cancer. The mechanisms underlying the activity of sericin against cancer cells are not fully understood. The present study investigated the effects of sericin on human colorectal cancer SW480 cells compared to normal colonic mucosal FHC cells. Since the size of the sericin protein may be important for its activity, two ranges of molecular weight were tested. Sericin was found to decrease SW480 and FHC cell viability. The small sericin had higher anti-proliferative effects than that of the large sericin in both cell types. Increased apoptosis of SW480 cells is associated with increased caspase-3 activity and decreased Bcl-2 expression. The anti-proliferative effect of sericin was accompanied by cell cycle arrest at the S phase. Thus, sericin reduced SW480 cell viability by inducing cell apoptosis via caspase-3 activation and down-regulation of Bcl-2 expression. The present study provides scientific data that support the protective effect of silk sericin against cancer cells of the colon and suggests that this protein may have significant health benefits and could potentially be developed as a dietary supplement for colon cancer prevention.     

E phage gene transfection associated to chemotherapeutic agents increases apoptosis in lung and colon cancer cells

The limited ability of conventional therapies to achieve the long-term survival of metastatic lung and colon cancer patients suggests the need for new treatment options. In this respect, genes encoding cytotoxic proteins have been proposed as a new strategy to enhance the activity of drugs, and combined therapies involving such genes and classical antitumoral drugs have been studied intensively. The E gene from phiX174 encodes a membrane protein with a toxic domain that leads to a decrease in tumour cell growth rates. Therefore, in order to improve the anti-tumour effects of currently used chemotherapeutic drugs on cancer cells, we investigated the association of the E suicide gene with these antineoplastic drugs. The E gene has antitumoral effects in both lung and colon cancer cells. In addition, expression of this gene induces ultrastructural changes in lung cancer transfected cells (A-549), although the significance of these changes remains unknown. The effect of combined therapy (gene and cytotoxic therapy) enhances the inhibition of tumour cell proliferation in comparison to single treatments. Indeed, our in vitro results indicate that an experimental therapeutic strategy based on this combination of E gene therapy and cytotoxic drugs may result in a new treatment strategy for patients with advanced lung and colon cancer.     

Acriflavine enhances radiosensitivity of colon cancer cells through endoplasmic reticulum stress-mediated apoptosis

Radiotherapy (RT) is one of the most effective tools in the clinical treatment of cancer. Because the tumor suppressor p53 plays a central role in radiation-mediated responses, including cell cycle-arrest and apoptosis, a number of studies have suggested that p53 could be a useful therapeutic target of anti-cancer agents. Accordingly, we sought to discover a new agent capable of increasing p53 activity. HCT116 colon cancer cells, containing wild-type p53, were stably transfected with a p53 responsive-luciferase (p53-Luc) reporter gene. A cell-based high-throughput screen of 7920 synthetic small molecules was performed in duplicate. Of the screened compounds, acriflavine (ACF) significantly increased p53-Luc activity in a concentration-dependent manner without causing toxicity. Pretreatment with ACF enhanced the induction of p53 protein expression and phosphorylation on serine 15 by γ-irradiation. Clonogenic assays showed that ACF pretreatment also potentiated radiation-induced cell death. The combination of irradiation and ACF treatment induced mitochondrial release of cytochrome c and significant activation of caspase-3 with PARP cleavage in colon cancer cells, demonstrating typical apoptotic cell death. Combined treatment with ACF and radiation increased the expression of Bax and Bad, while decreasing expression of Bcl-2. In addition, the ACF/radiation treatment combination induced endoplasmic reticulum (ER) stress responses mediated by IRE1α (inositol-requiring transmembrane kinase and endonuclease 1α), eIF-2α (eukaryotic initiation factor 2α), caspase-2/12, and CHOP (C/EBP homologous protein). The knockdown of IRE1α by siRNA inhibited the apoptotic cell death induced by ACF/radiation treatment. In vivo studies showed that combined treatment with ACF and radiation significantly inhibited the growth of tumors in colorectal cancer xenografted mice. These results indicate that ACF acts through p53-dependent mitochondrial pathways and ER stress signals, and could be a promising radiosensitizer.