No abstract

Prostate cancer is one of the leading causes of death in men aged 40 to 55. Genistein isoflavone (4′, 5′, 7‐trihydroxyisoflavone) is a dietary phytochemical with demonstrated anti‐tumour activities in a variety of cancers. Topotecan Hydrochloride (Hycamtin) is an FDA‐approved chemotherapy drug, primarily used for secondary treatment of ovarian, cervical and small cell lung cancers. This study was to demonstrate the potential anticancer efficacy of genistein‐topotecan combination in LNCaP prostate cancer cells and the mechanism of the combination treatment. The LNCaP cells were grown in complete RPMI medium, and cultured at 37°C, 5% CO2 for 24–48 hrs to achieve 70–90% confluency. The cells were treated with varying concentrations of genistein, topotecan and genistein‐topotecan combination and incubated for 24 hrs. The treated cells were assayed for (i) post‐treatment sensitivity using MTT assay and DNA fragmentation, (ii) treatment‐induced apoptosis using caspase‐3 and ‐9 binding assays and (iii) treatment‐induced ROS generation levels. The overall data indicated that (i) both genistein and topotecan induce cellular death in LNCaP cells, (ii) genistein‐topotecan combination was significantly more efficacious in reducing LNCaP cell viability compared with either genistein or topotecan alone, (iii) in all cases, cell death was primarily through apoptosis, via the activation of caspase‐3 and ‐9, which are involved in the intrinsic pathway, (iv) ROS generation levels increased significantly with the genistein‐topotecan combination treatment. Treatments involving genistein‐topotecan combination may prove to be an attractive alternative phytotherapy or adjuvant therapy for prostate cancer.

C-phycocyanin (C-PC) from Spirulina has been previously shown to have anticancer properties. Here, we report on anticancer activity of C-PC that was isolated from the novel cyanobacterium Limnothrix sp. 37-2-1. C-PC from this organism exhibited anticancer properties in our in vitro systems; however, the required doses were well above the range of anticancer drugs normally used. Therefore, we conducted several experiments to test whether lower-than-usual doses of the anticancer drug topotecan (TPT) can offer the same level of cytotoxic effects as normal doses when combined with C-PC. For this purpose, cytotoxicities of C-PC and TPT were tested using the LNCaP (prostate cancer) cells. We found that when only 10% of a typical dose of TPT was combined with C-PC, the cancer cells were killed at a higher rate than when TPT was used alone at full dose. Similarly, we were also able to detect an increased level of radical oxygen species (ROS) generation as well as an increase in activities of caspase-9 and caspase-3 when these two compounds were used in combination. Taken together, our findings suggest that combining C-PC from Limnothrix sp. with the lower dose of TPT can induce apoptosis through generation of ROS and activation of caspases. In that respect, we suggest that C-PC can potentially improve the efficacy of the currently available anticancer drug, and therefore diminish its harsh side effects in the patient.

PC12 cells express two atrial-natriuretic-factor-(ANF)-receptor subtypes with molecular masses of 130,000 (B receptor) and 70,000 (C receptor). The B-receptor subtype constitutes 65% of the cell-surface receptor population, and the remaining 35% are C receptors as determined by saturation binding studies in the presence of C-ANF, a C-receptor-selective analogue. ANF-(99-126)-peptide [ANF(99-126)], which can bind to both B- and C-receptor subtypes, was rapidly internalized into the cells after incubation at 37 degrees C. Internalization of 125I-ANF(99-126) was used as an index of the receptor-mediated endocytosis and to quantify receptor internalization. In the presence of a saturating concentration of C-ANF, receptor-mediated internalization of 125I-ANF(99-126) was reduced by 24%, indicating B receptor mediate 76% of ligand internalization. Incubation of cells with 10 microM-ANF at 37 degrees C down-regulated both receptor subtypes as reflected by decreased surface binding. Time-dependent studies suggest that B- and C-receptor subtypes undergo differential down-regulation. Incubation of down-regulated cells for 120 min in ANF-free medium produced a recovery of 35% of the original cell-surface binding. Affinity cross-linking of 125I-ANF to the receptors on the plasma membrane in re-incubated (up-regulated) cells demonstrated expression of predominantly the B-receptor subtype. Monensin blocked 72% of receptor up-regulation, whereas cycloheximide inhibited 43%, suggesting an active recycling mechanism involved in mediating up-regulation of the B receptors. The present study demonstrates a rapid internalization and intracellular recycling mechanism for B receptors in PC12 cells. C receptors also undergo internalization and down-regulation, but recycling of this receptor subtype into the plasma membrane occurs at a lower rate and to a lesser extent than is the case for the B receptor.

Bromelain is a proteolytic enzyme extracted from the stems and the immature fruits of pineapple that was found to be antitumorigenic in different in vitro models. Bromelain has been reported to promote apoptosis, particularly in breast cancer cells, with the up-regulation of c-Jun N-terminal kinase and p38 kinase. Our study was designed to determine if bromelain could induce apoptosis in GI-101A breast cancer cells. GI-101A cells were treated with increasing concentrations of bromelain for 24 hours. The effect of bromelain for inducing cell death via activation of the apoptosis mechanism in GI-101A cells was further determined by using caspase-9 and caspase-3 assays along with the M30-Apoptosense assay to measure cytokeratin 18 (CK18) levels in the cytoplasm of the cultured cancer cells. A dose-dependent increase in the activities of caspase-9 and caspase-3 coinciding with elevation of CK18 levels was found in bromelain-treated cells compared with control cells. Furthermore, the apoptosis induction by bromelain was confirmed by DNA fragmentation analysis and 4,6'-diamino-2-phenylindole dihydrochloride fluorescence staining of the nucleus. Our results indicate an increase in apoptosis-related cell death in breast cancer cells with increasing concentrations of bromelain.