bafilomycin-a1 and Adenocarcinoma

Maple syrup is a natural sweetener consumed worldwide. Active ingredients of maple syrup possess antitumor effects; however, these ingredients are phenolic compounds. The present study aimed to investigate components other than phenolic compounds that may have antitumor effects against colorectal cancer (CRC). Cell proliferation assays demonstrated that treatment with the more than 10,000 molecular weight fraction significantly inhibited viability in DLD‑1 cells. Therefore, we hypothesized that the protein components of maple syrup may be the active ingredients in maple syrup. We obtained protein components from maple syrup by ammonium sulfate precipitation, and treatment with the protein fraction of maple syrup (MSpf) was found to exhibit a potential antitumor effect. MSpf‑treated DLD‑1 colon adenocarcinoma cells exhibited significantly decreased proliferation, migration and invasion. In addition, upregulation of LC3A and E‑cadherin and downregulation of MMP‑9 expression levels were observed following MSpf treatment. Investigation of the components of MSpf suggested that it was primarily formed of advanced glycation end products (AGEs). Therefore, whether AGEs in MSpf affected the STAT3 pathway through the binding to its receptor, receptor of AGE (RAGE), was assessed. MSpf treatment was associated with decreased RAGE expression and STAT3 phosphorylation. Finally, to determine whether autophagy contributed to the inhibitory effect of cell proliferation following MSpf treatment, the effect of MSpf treatment on autophagy induction following bafilomycin A1 treatment, a specific autophagy inhibitor, was assessed. The inhibitory effect of MSpf treatment on cell proliferation was enhanced through the inhibition of autophagy by bafilomycin A1 treatment. These results suggested that AGEs in MSpf suppressed cell proliferation and epithelial‑mesenchymal transition through inhibition of the STAT3 signaling pathway through decreased RAGE expression. Therefore, AGEs in MSpf may be potential compounds for the development of antitumor drugs for the treatment of CRC with fewer adverse effects compared with existing antitumor drugs.

Topics: Acer; Adenocarcinoma; Colonic Neoplasms; Glycation End Products, Advanced; Humans

The pyrrolo-1,5-benzoxazepines (PBOXs) are a novel group of selective apoptotic agents displaying promising therapeutic potential in both ex vivo chemotherapy-refractory patient samples and in vivo murine carcinoma models. In this report, we present novel data concerning the induction of autophagy by the PBOXs in adenocarcinoma-derived colon cancer cells. Autophagy is a lysosome-dependent degradative pathway recently associated with chemotherapy. However, whether autophagy facilitates cell survival in response to chemotherapy or contributes to chemotherapy-induced cell death is highly controversial. Autophagy was identified by enhanced expression of LC3B-II, an autophagosome marker, an increase in the formation of acridine orange-stained cells, indicative of increased vesicle formation and electron microscopic confirmation of autophagic structures. The vacuolar H+ ATPase inhibitor bafilomycin-A1 (BAF-A1) inhibited vesicle formation and enhanced the apoptotic potential of PBOX-6. These findings suggest a cytoprotective role of autophagy in these cells following prolonged exposure to PBOX-6. Furthermore, BAF-A1 and PBOX-6 interactions were determined to be synergistic and caspase-dependent. Potentiation of PBOX-6-induced apoptosis by BAF-A1 was associated with a decrease in the levels of the anti-apoptotic protein, Mcl-1. The data provide evidence that autophagy functions as a survival mechanism in colon cancer cells to PBOX-6-induced apoptosis and a rationale for the use of autophagy inhibitors to further enhance PBOX‑6‑induced apoptosis in colon cancer.

Topics: Adenocarcinoma; Animals; Apoptosis; Autophagy; Caspases; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Drug Synergism; Humans; Lysosomes; Macrolides; Mice; Microtubule-Associated Proteins; Oxazepines; Pyrroles

Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Autophagy; BH3 Interacting Domain Death Agonist Protein; Caspase 10; Caspase 3; Caspase 8; Caspase Inhibitors; Cell Line, Tumor; Colonic Neoplasms; Humans; Macrolides; Oligopeptides; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; Silybin; Silymarin; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

Topics: Adenocarcinoma; Animals; Autophagy; Cell Line, Tumor; Cell Proliferation; Chloroquine; Humans; Immunoglobulin G; Macrolides; Melphalan; Mice; Oxidative Phosphorylation; Pancreatic Neoplasms; Reactive Oxygen Species; Xenograft Model Antitumor Assays

Microcalcifications containing calcium hydroxyapatite (HA) are often associated with malignant human breast lesions. Frequently, they are the only mammographic features that indicate the presence of a tumoural lesion. We previously reported the induction of both mitogenesis and prostaglandin E2 (PGE2) production and the increased activities of matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in normal human mammary epithelial cells and breast cancer cell lines, treated with HA. In the present study we attempted to elucidate the mechanism of these biological effects. Firstly, we found that direct cell-crystal contact was required for induction of mitogenesis as the effect was not merely a result of isotopic exchange of calcium into the culture medium. Treatment with bafilomycin A1, a proton pump inhibitor, abrogated HA-induced mitogenesis to control cell levels. These results suggest that phagocytosis and intracellular crystal dissolution is required for HA-induced mitogenesis. We also demonstrated that the increase in prostaglandin E2, previously reported, is due, at least in part, to HA-induced upregulation of cyclooxygenase-2 (COX-2) in Hs578T cells. An accumulation of MMP-1 mRNA was also shown in response to HA stimulation in Hs578T cells. Furthermore, a HA-induced increase in interleukin-1beta (IL-1beta), a potent inducer of MMP-1 gene expression, was demonstrated in Hs578T cells at 2 and 4 h. Treatment with phosphocitrate (PC) (a naturally occurring inhibitor of calcium phosphate crystallisation, which is known to block a number of HA-induced biological effects in other cell types) blocked HA-mediated mitogenesis, as well as, COX-2, MMP-1 and IL-1beta induction, at the transcriptional level. These results show that calcium HA crystals are capable of exerting significant biological effects on surrounding cells which can be abrogated by PC and emphasise the role of calcium HA in amplifying the pathological process involved in breast cancer.

Topics: Adenocarcinoma; Anti-Bacterial Agents; Breast Neoplasms; Calcinosis; Cell Division; Citrates; Cyclooxygenase 2; Dinoprostone; Durapatite; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1; Isoenzymes; Macrolides; Matrix Metalloproteinase 1; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Tumor Cells, Cultured; Up-Regulation

We investigated the effects of vacuolating cytotoxin (VacA) prepared from Helicobacter pylori on the metabolism of gastric epithelial cells, AZ-521. VacA caused the ATP levels to decrease in a time-dependent manner; by approximately 20% in 6 h, 35% in 12 h and 50% in 24 h, at a concentration of 120 nM. This decrease was also dependent on the concentration of VacA. To evaluate the impairment of mitochondria by VacA, mitochondrial membrane potential was estimated by flow cytometric analysis using 3, 3'-dihexyloxacarbocyanine iodide as a substrate. VacA decreased membrane potential with the relative fluorescence intensity of AZ-521 cells in 6 h from 52+/-3 to 24+/-1. Treatment of the cells with bafilomycin A1, a specific inhibitor of vacuolar ATPase proton pump, showed no apparent effect on these changes in the levels of ATP and the mitochondrial membrane potential. Secondly, we estimated the effect of VacA on oxygen consumption. VacA inhibited oxygen consumption in AZ-521 cells: the levels of PO 2 in the medium of control cells decreased by 73% in 3 h and 37% in 6 h, whereas those in VacA-treated cells were 84% in 3 h and 59% in 6 h. Flow cytometric analysis showed the number of cells in the G0/G1 phase was increased by VacA. Taken together, VacA induced an inactivation of energy metabolism followed by mitochondrial damage, leading to impairment of the cell cycle in gastric epithelial cells.

Topics: Adenocarcinoma; Adenosine Triphosphate; Anti-Bacterial Agents; Bacterial Proteins; Bacterial Toxins; Cell Cycle; Cell Respiration; Cytotoxins; Enzyme Inhibitors; Flow Cytometry; Gastric Mucosa; Helicobacter pylori; Humans; Macrolides; Membrane Potentials; Mitochondria; Oxygen Consumption; Stomach Neoplasms; Tumor Cells, Cultured

Bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, can inhibit the growth of a variety of cultured cells in a dose-dependent manner, but its mechanism is unclear. The aim of this study was to examine whether bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis. The effect of bafilomycin A1 on tumour growth in vitro and in vivo was examined using an MTT assay and an in vivo tumour model. The presence or absence of apoptosis was determined by morphology and DNA analysis of tumour cells. The concentration of bafilomycin A1 for 50 per cent inhibition of cell viability during 72 h by the MTT assay was 5 nm. In DNA analysis, a ladder of fragmented DNA was detected in Capan-1 cells treated with bafilomycin A1 at concentrations greater than 10 nm for 24 h. Nude mice bearing a xenografted Capan-1 cell line tumour received 4 weeks of bafilomycin A1 (1.0 mg/kg per day). This treatment significantly inhibited tumour growth compared with controls after 21 days (P < 0.05). Histopathological examination of tumour cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis.

Topics: Adenocarcinoma; Adult; Animals; Anti-Bacterial Agents; Apoptosis; DNA Fragmentation; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Enzyme Inhibitors; Humans; Macrolides; Male; Mice; Mice, Inbred BALB C; Pancreatic Neoplasms; Proton-Translocating ATPases; Tumor Cells, Cultured