Molecular mechanism of C-phycocyanin induced apoptosis in LNCaP cells.

The usefulness of Marine-derived products as the source of anticancer agents has been explored for many decades. The objective of our study was to investigate the molecular mechanism by which C-PC induces apoptosis in monotherapy as well as in combination treatment with a known chemotherapeutic drug named Topotecan (TPT) using prostate cancer cells (LNCaP). To determine the intracellular mechanism of action, we analyzed the gene expression profile of C-PC treated cells using human apoptosis RT2 profiler PCR array, which indicated that C-PC was able to regulate both anti- and pro-apoptotic genes significantly. Detailed analysis revealed increases in the levels of Bax, Apaf-1 (pro-apoptotic proteins) along with the activation of the key apoptotic proteases such as caspase-8, caspase-9, and caspase-3. Similarly, analysis of anti-apoptotic proteins demonstrated a decrease in the expression of Bcl-2, Mcl-1, and survivin. Results from the whole-cell incubation studies indicated that C-PC was only binding to the plasma membrane-associated receptor proteins. LNCaP cells treated with C-PC alone and in combination with TPT showed increased expression of the death receptor FAS (also known as FAS or CD95) along with cleaved PARP, confirming its importance. Our study is significant since it is providing greater insight into the apoptotic mechanisms triggered by C-PC as well as emphasizing the involvement of FAS in mediating its effects. Furthermore, our results with combination treatments suggest that-PC could improve the anticancer effects of drugs such as TPT that are currently used for cancer treatments. In addition, use of C-PC in combination can also diminish the side effects resulting from conventional chemotherapeutic agents such as TPT.

Identification of a new tamoxifen-xanthene hybrid as pro-apoptotic anticancer agent.

Breast cancer is the most diagnosed type of cancer among women for which an exhaustive cure has not been discovered yet. Nowadays, tamoxifen still represents the gold standard for breast cancer therapy; it acts on both estrogen receptor-positive and estrogen receptor-negative breast cancers. Unfortunately, its toxicity and the related chemoresistance undermine its antitumor potential. In this paper, new tamoxifen-based derivatives with a rigid structural motif in their structure were designed, synthesized, and evaluated to assess their antitumor behavior. All the tested compounds affected estrogen receptor-positive tumor (MCF-7) cell growth, even with different extents, among which, the most active ones proved also to induce mitochondria-mediated apoptosis through activation of PARP cleavage, decrease in Bax/Bcl-2 ratio and increase in Bim gene expression levels. Here we found that the compound 1, carrying a rigid xanthene core, turned out to be the most promising of the set showing an activity profile comparable to that of tamoxifen. Furthermore, a more favorable genotoxic profile than tamoxifen made compound 1 a promising candidate for further studies.

Cyproterone acetate enhances TRAIL-induced androgen-independent prostate cancer cell apoptosis via up-regulation of death receptor 5.

BACKGROUND: Virtually all prostate cancer deaths occur due to obtaining the castration-resistant phenotype after prostate cancer cells escaped from apoptosis and/or growth suppression initially induced by androgen receptor blockade. TNF-related apoptosis-inducing ligand (TRAIL) was an attractive cancer therapeutic agent due to its minimal toxicity to normal cells and remarkable apoptotic activity in tumor cells. However, most localized cancers including prostate cancer are resistant to TRAIL-induced apoptosis, thereby creating a therapeutic challenge of inducing TRAIL sensitivity in cancer cells. Herein the effects of cyproterone acetate, an antiandrogen steroid, on the TRAIL-induced apoptosis of androgen receptor-negative prostate cancer cells are reported. METHODS: Cell apoptosis was assessed by both annexin V/propidium iodide labeling and poly (ADP-ribose) polymerase cleavage assays. Gene and protein expression changes were determined by quantitative real-time PCR and western blot assays. The effect of cyproterone acetate on gene promoter activity was determined by luciferase reporter assay. RESULTS: Cyproterone acetate but not AR antagonist bicalutamide dramatically increased the susceptibility of androgen receptor-negative human prostate cancer PC-3 and DU145 cells to TRAIL-induced apoptosis but no effects on immortalized human prostate stromal PS30 cells and human embryonic kidney HEK293 cells. Further investigation of the TRAIL-induced apoptosis pathway revealed that cyproterone acetate exerted its effect by selectively increasing death receptor 5 (DR5) mRNA and protein expression. Cyproterone acetate treatment also increased DR5 gene promoter activity, which could be abolished by mutation of a consensus binding domain of transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP) in the DR5 gene promoter. Cyproterone acetate increases CHOP expression in a concentration and time-dependent manner and endoplasmic reticulum stress reducer 4-phenylbutyrate could block cyproterone acetate-induced CHOP and DR5 up-regulation. More importantly, siRNA silencing of CHOP significantly reduced cyproterone acetate-induced DR5 up-regulation and TRAIL sensitivity in prostate cancer cells. CONCLUSIONS: Our study shows a novel effect of cyproterone acetate on apoptosis pathways in prostate cancer cells and raises the possibility that a combination of TRAIL with cyproterone acetate could be a promising strategy for treating castration-resistant prostate cancer.

A novel L-fucose-binding lectin from Fenneropenaeus indicus induced cytotoxicity in breast cancer cells.

Lectins are omnipresent in almost all life forms, being the proteins which specifically bind to carbohydrate moieties on the cell surface; they have been explored for their anti-tumour activities. In this study, we purified a fucose specific-lectin (IFL) from Fenneropenaeus indicus haemolymph using fucose-affinity column and characterized for its haemagglutination activity, carbohydrate specificity, dependency on cations and cytotoxicity against cancer cells. The lectin showed non-specificity against human erythrocytes. It was a Ca2+-dependent lectin which remained stable over wide pH and temperature ranges. The lectin showed effective dose dependent cytotoxicity against different human cancer cell lines and induced apoptosis in MCF-7 cells as evidenced by DNA ladder assay and PARP cleavage in a dose dependent manner. Moreover, an increased p21 level corresponding to cyclin D downregulation in response to IFL treatment was observed which might work as probable factors to inhibit cell growth and induce apoptosis of MCF-7 cells. Therefore, we report a novel lectin from the prawn haemolymph with high specificity for L-fucose and antiproliferative towards human cancer cells. However, further establishment of the modus operandi of this lectin is required to enable its biotechnological applications.

Methyleugenol and selected oxidative metabolites affect DNA-Damage signalling pathways and induce apoptosis in human colon tumour HT29 cells.

Previously the food carcinogen methyleugenol was found to be cytotoxic and genotoxic in multiple cell lines and in primary hepatocytes. In this study, the question addressed was whether methyleugenol and the selected oxidative metabolites, 1'-hydroxymethyleugenol, methyleugenol-2',3'-epoxide and 3'-oxomethylisoeugenol trigger a DNA damage response in the human colon carcinoma HT29 cell line. Most notably investigations by flow cytometry revealed that the metabolites induce an accumulation of HT29 cells in the G2 phase of the cell cycle. DNA damage response is characterised by a time-delayed phosphorylation of ATM (ataxia-telangiectasia, mutated)/ATR (ATM- and Rad3-related) kinases and checkpoint kinase 1 after 2 h of incubation, and the tumour suppressor protein p53 only after 24 h of incubation. The test compounds induced apoptotic cell death indicated by cleavage of caspase 3 and poly-(ADP-ribose)-polymerase after a prolonged incubation time up to 72 h. In addition, activation of ATM/ATR-signalling cascade might contribute to apoptosis induction to a certain extent. However, clarification of this relationship awaits experimental confirmation.

Hypoxia-activated cytotoxicity of benznidazole against clonogenic tumor cells.

Solid tumors contain numerous regions with insufficient oxygen concentrations, a condition termed hypoxia. Tumor hypoxia is significantly associated with metastasis, refractory to conventional cancer therapies, and poor patient survival. Therefore, eradication of hypoxic tumor cells will likely have significant impact on the overall progression-free patient survival. This article reports a new discovery that Benznidazole, a bioreductive drug currently used to treat Chagas disease caused by the parasitic protozoan Trypanosoma cruzi, is activated by hypoxia and can kill clonogenic tumor cells especially those under severe hypoxic conditions (≤0.1 % O2). This type of hypoxia selectivity is important in that severely hypoxic tumor microenvironment is where tumor cells exhibit the strongest resistance to therapy. Mechanistically, activation of Benznidazole coincides with the stabilization of the Hypoxia-Inducible Factor 1α (HIF-1α), suggesting that Benznidazole is activated after tumor cells have entered into a fully hypoxic state. Under such hypoxic conditions, Benznidazole induces the formation of 53BP1 foci, a hallmark of DNA double-stranded breaks that can cause clonogenic inhibition or cell death. These results demonstrate that Benznidazole is a hypoxia-activated cytotoxin with the potential to specifically eliminate hypoxic tumor cells.

Apoptosis Induction by Ocimum sanctum Extract in LNCaP Prostate Cancer Cells.

Tulsi (Ocimum sanctum Linn), commonly known as "holy basil," has been used for the treatment of a wide range of ailments in many parts of the world. This study focuses on apoptosis-inducing ability of tulsi extract on prostate cancer cells. For this purpose LNCaP prostate cancer cells were treated with different concentrations of 70% ethanolic extract of tulsi (EET) and then the cytotoxicity was determined after 24 and 48 h. After treatment with EET externalization of phosphatidyl serine (PS) from the inner membrane to outer leaflet of the plasma membrane was clearly evidenced by the results obtained from both flow cytometry analysis with Annexin V-FITC and pSIVA-IANBD binding fluorescence microscopy assay. Depolarization of the mitochondrial membrane potential was also evidenced by the presence of 5,5',6,6'-tetrachlolo-1,1',3,3'-tetraethyl benzimedazolyl carbocyanine iodide (JC-1) monomeric form in the EET-treated cells that emitted the green fluorescence when compared with the control cells that emitted the red fluorescence due to aggregation of JC-1. Furthermore, the level of poly (ADP-ribose) polymerase (PARP) cleavage and Bcl-2 were determined using western blot analysis. When compared to the control cells the level of cleaved PARP was found to be higher with a concomitant decrease in the Bcl-2 level after 24 h of treatment of cells with EET. In addition, treatment with EET significantly elevated the activities of caspase-9 and caspase-3 in LNCaP cells compared with the control. Also, after 48 h of treatment all doses used in this study showed clear fragments of DNA, which is one of the hallmarks of apoptosis. Taken together, our findings suggest that, EET can effectively induce apoptosis in LNCaP cells via activation of caspase-9 and caspase-3 that can eventually lead to DNA fragmentation and cell death.

Induction of apoptosis by doxorubicin-transferrin conjugate compared to free doxorubicin in the human leukemia cell lines.

In our research we compared the effect of doxorubicin (DOX) and doxorubicin-transferrin (DOX-TRF) conjugate on the induction of programmed cell death. All experiments were carried out on human leukemia cells: CCRF-CEM, K562 sensitive and resistant to DOX, (K562/DOX), which are the molecular model for the chronic and acute form of hematological malignancies, respectively. At the same time, studies were also performed on normal, peripheral blood mononuclear cells (PBMCs). The first stages of apoptosis, connected with externalization of phosphatidylserine (PS), were evaluated after comparing the viability of tested cell lines treated with DOX-TRF conjugate or free DOX. Morphological changes of nuclei connected with apoptosis were analyzed by double staining Hoechst 33258/propidium iodide. Subsequently, we conducted a more accurate evaluation of DOX-TRF-trigged cell death by using DNA ladder assay, measuring the activation of caspase-3, -8 and -9 and changes in poly-ADP ribose polymerase (PARP) activity. The percentage of apoptotic cells reached its maximum at 24 and 48 h incubation. Prolonged treatment time with DOX-TRF conjugate progressively increased the level of necrotic cells. At 24-48 h time points, we observed a significant increase in the activity of apoptosis-characterized enzymes (caspases -8, -9, -3). This study provided the evidence that DOX-TRF conjugate triggers apoptotic pathway connected with DNA damage mediated by the activation of pro-caspases and PARP cleavage.