benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Esophageal-Neoplasms

Oesophageal cancer is the ninth most common cancer in the world and the second most common cancer among South African men. It also has one of the lowest possibilities of cure, with the 5-year survival rate estimated to be only 10% overall. Sutherlandia frutescens, or the "cancer bush", is a medicinal plant indigenous to southern Africa that is believed to have anti-cancer and anti-proliferative properties. The aim of this study was to investigate the potential apoptosis-inducing effects of two S. frutescens extracts and one Sutherlandia tomentosa extract on the SNO oesophageal cancer cell line.. Cell viability and morphology of SNO cells were evaluated following exposure to the extracts. Apoptotic markers including cytochrome c translocation and phosphatidylserine externalisation were quantified by flow cytometry. The activity of caspases 3 and 7 was evaluated with spectrofluorometry. Apoptosis was evaluated in the presence of the pan-caspase inhibitor, Z-VAD-fmk. The effect of the extracts was compared to non-cancerous peripheral blood mononuclear cells (PBMCs).. Time- and dose-response studies were conducted to establish treatment conditions of 2.5 and 5mg/ml of crude plant extracts. Microscopy studies revealed that S. frutescens- and S. tomentosa-treated SNO cells had morphological features characteristic of apoptosis. Annexin V/propidium iodide flow cytometry confirmed that the extracts do, in fact, induce apoptosis in the SNO cells. Caspase inhibition studies seem to indicate that extracts A (S. frutescens (L.) R. Br. subsp. microphylla from Colesberg), B (S. frutescens (L.) R. Br. subsp. microphylla from Platvlei) and C (S. tomentosa Eckl. & Zeyh from Stil Bay) are able to induce caspase-dependent as well as -independent cell death. The S. frutescens and S. tomentosa extracts were found to be more cytotoxic to cancerous SNO cells when compared to the PBMCs.. S. frutescens and S. tomentosa extracts show promise as apoptosis-inducing anti-cancer agents.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspase 7; Caspase Inhibitors; Cell Line, Tumor; Cell Shape; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Esophageal Neoplasms; Fabaceae; Flow Cytometry; Humans; Leukocytes, Mononuclear; Phosphatidylserines; Plant Extracts; Plants, Medicinal; Protein Transport; Spectrometry, Fluorescence; Time Factors

Fascin induces membrane protrusions and cell motility. Fascin overexpression was associated with poor prognosis, and its downregulation reduces cell motility and invasiveness in esophageal squamous cell carcinoma (ESCC). Using a stable knockdown cell line, we revealed the effect of fascin on cell growth, cell adhesion and tumor formation.. We examined whether fascin is a potential target in ESCC using in vitro and in vivo studies utilizing a specific siRNA. We established a stable transfectant with downregulated fascin from KYSE170 cell line.. The fascin downregulated cell lines showed a slower growth pattern by 40.3% (p < 0.01) and detachment from collagen-coated plates by 53.6% (p < 0.01), compared to mock cells, suggesting that fascin plays a role in cell growth by maintaining cell adhesion to the extracellular matrix. In vivo, the tumor size was significantly smaller in the tumor with fascin knockdown cells than in mock cells by 95% at 30 days after inoculation.. These findings suggest that fascin overexpression plays a role in tumor growth and progression in ESCC and that cell death caused by its downregulation might be induced by cell adhesion loss. This indicates that targeting fascin pathway could be a novel therapeutic strategy for the human ESCC.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Carcinoma, Squamous Cell; Carrier Proteins; Caspase Inhibitors; Caspases; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Collagen; Cysteine Proteinase Inhibitors; Esophageal Neoplasms; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Microfilament Proteins; RNA Interference; RNA, Small Interfering; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

To study the molecular mechanism of TAp63gamma-induced cell apoptosis.. Transcription and protein expression of apoptosis inducing factor and p63 were investigated by immunohistochemistry and RT-PCR in human esophageal squamous carcinoma cell line EC9706 respectively. Twenty-four hours after transfection with pcDNA3.1-TAp63gamma, the apoptosis and translocation of apoptosis inducing factor in EC9706 cells were studied by flow cytometry, laser confocal microscopy and mitochondrial/cytosol/nuclear extraction analysis respectively. Down-regulation of apoptosis inducing factor protein was achieved by RNAi and pretreatment with caspase inhibitor zVAD.fmk of EC9706 cells.. Presence of protein expressions of apoptosis inducing factor and absence of TAp63gamma was observed in the cytoplasm of untransfected cells. RT-PCR verified the subtype of p63 in EC9706 cells was DeltaNp63. After 24 hours of transfection, both nuclear and cytoplasmic expression of apoptosis inducing factor protein were observed in cells transfected with TAp63gamma and p53 expression vectors, but not in cells transfected with control vector. Cell apoptosis rates were 1.37%, 13.64%, 4.52%, 4.03% and 1.91% in the pcDNA3.1 transfection group, pcDNA3.1-TAp63gamma transfection group, apoptosis inducing factor siRNA and pcDNA3.1-TAp63gamma transfection group, zVAD.fmk treatment group, and the group receiving apoptosis inducing factor siRNA, plus zVAD.fmk treatment and pcDNA3.1-TAp63gamma transfection, respectively.. Apoptosis inducing factor of EC9706 cells is released from mitochondria into both the cytoplasm and nucleus during TAp63gamma induced apoptosis. Down-regulation of apoptosis inducing factor inhibits TAp63gamma-induced apoptosis. Overall, TAp63gamma-induced apoptosis is dependent on the expression of apoptosis inducing factor and caspase.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Inducing Factor; Carcinoma, Squamous Cell; Caspase Inhibitors; Cell Line, Tumor; Cell Nucleus; Cytoplasm; Down-Regulation; Esophageal Neoplasms; Humans; Mitochondria; Plasmids; Protein Transport; RNA Interference; RNA, Small Interfering; Trans-Activators; Transcription Factors; Transfection; Tumor Suppressor Proteins