benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Uterine-Cervical-Neoplasms

4-(3',3'-Dimethylallyloxy)-5-methyl-6-methoxy-phthalide (DMMP) has previously been isolated from the endophytic fungus Pestalotiopsis photiniae. Although the cytotoxic activities of DMMP have been reported, little is known concerning the molecular mechanism of its cytotoxic effect. In the present study, we investigated the effect of DMMP on the growth of several types of cancer cell lines and investigated the mechanism of its antiproliferative effect. DMMP caused the growth inhibition of human cancer lines HeLa, MCF7 and MDA-MB-231, but had little antiproliferative effect on MRC5 normal lung cells. DMMP also significantly caused cell cycle arrest in the G1 phase and upregulated the cyclin-dependent kinase inhibitor p27KIPI protein in the HeLa cells. Moreover DMMP was able to induce marked nuclear apoptotic morphology in HeLa cells. DMMP induced apoptosis and loss of mitochondrial membrane potential (ΔΨm) in the HeLa cells. Although the activated forms of caspase-9 and -3 in HeLa cells were detected, pretreatment with caspase inhibitors (Ac-DEVD-CHO and Z-VAD-FMK) failed to attenuate DMMP-induced cell death. In addition, protein levels of the p53 family members, p53 and p73, were upregulated, and DMMP significantly increased the mRNA expression of pro-apoptotic Bcl-2 family genes (PUMA, NOXA, Bax, Bad and Bim). HPV E6-E7 mRNA levels were reduced. In conclusion, DMMP demonstrates potential for use in the treatment of cervical cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-Associated Death Protein; Benzofurans; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p27; DNA-Binding Proteins; Female; G1 Phase Cell Cycle Checkpoints; HeLa Cells; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondria; Nuclear Proteins; Oligopeptides; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Up-Regulation; Uterine Cervical Neoplasms; Xylariales

T-2 toxin is the most toxic trichothecene and both humans and animals suffer from several pathological conditions after consumption of foodstuffs contaminated with trichothecenes. We investigated the molecular mechanism of T-2 toxin induced cytotoxicity and cell death in HeLa cells. T-2 toxin at LC50 of 10 ng/ml caused time dependent increase in cytotoxicity as assessed by dye uptake, lactatedehydrogenase leakage and MTT assay. The toxin caused generation of reactive oxygen species as early as 30 min followed by significant depletion of glutathione levels and increased lipid peroxidation. The results indicate oxidative stress as underlying mechanism of cytotoxicity. Single stranded DNA damage after T-2 treatment was observed as early as 2 and 4h by DNA diffusion assay. The cells exhibited apoptotic morphology like condensed chromatin and nuclear fragmentation after 4h of treatment. Downstream of T-2 induced oxidative stress and DNA damage a time dependent increase in expression level of p53 protein was observed. The increase in Bax/Bcl2 ratio indicated shift in response, in favour of apoptotic process in T-2 toxin treated cells. Western blot analysis showed increase in levels of mitochondrial apoptogenic factors Bax, Bcl-2, cytochrome-c followed by activation of caspases-9, -3 and -7 leading to DNA fragmentation and apoptosis. In addition to caspase-dependent pathway, our results showed involvement of caspase-independent AIF pathway in T-2 induced apoptosis. Broad spectrum caspase inhibitor z-VAD-fmk could partially protect the cells from DNA damage but could not inhibit AIF induced oligonucleosomal DNA fragmentation beyond 4 h. Results of the study clearly show that oxidative stress is the underlying mechanism by which T-2 toxin causes DNA damage and apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Cell Survival; DNA Damage; DNA, Neoplasm; Female; Glutathione; HeLa Cells; Humans; Lipid Peroxidation; Malondialdehyde; Mitochondria; Neuroprotective Agents; Oxidative Stress; Signal Transduction; T-2 Toxin; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Cervical carcinomas are most frequently associated with human papillomaviruses (HPV), whose E6 and E7 oncogenes products induce cellular immortalization. The papillomavirus E2 protein is a transcription factor, which represses the expression of the viral oncogenes, and activates viral DNA replication during the vegetative viral cycle. This protein is specifically inactivated in HPV18-associated carcinoma cells, suggesting that E2 functions prevent carcinogenic progression. Indeed, ectopic expression of E2 in cervical carcinoma cells strongly inhibits cell proliferation. Here we show that above a threshold level of expression, the E2 protein induces apoptosis, independently of other viral functions. The amino-terminal domain is responsible for this apoptotic activity, but surprisingly with no involvement of its transcriptional functions. The death pathway triggered by E2 relies on activation of the initiator caspase 8, specific of the extrinsic pathway of apoptosis. E2 itself is cleaved by caspases during cell death, providing an example of an apoptotic inducer that is itself a target for caspase processing. The autonomous proapoptotic activity of HPV18 E2 described here may counteract the proliferative functions of viral oncogenes, and renders the inactivation of E2 crucial for carcinogenic progression.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cells, Cultured; Cysteine Proteinase Inhibitors; Female; Humans; Mutation; Oncogene Proteins, Viral; Protein Structure, Tertiary; Recombinant Proteins; Threshold Limit Values; Trans-Activators; Transcription, Genetic; Uterine Cervical Neoplasms

Extract of Coprinus disseminatus (pers. Fr.) (C. disseminatus) culture broth (EDCB) inhibits proliferation and induces apoptosis in the human cervical carcinoma cells at 5 microg/ml. To determine whether the cell death induced by the EDCB recruits caspases or not, one of the exclusive pathways in cell death, we examined caspase-3 activity in this cell death process. The activity of caspase-3 was remarkably increased when the cell was treated with EDCB, and this activity was nullified by Z-VAD-FMK, a well known caspase-3 inhibitor. From these results, we would expect the EDCB to contain substances with the ability to induce apoptosis in the human cervical carcinoma cells. The extent of the EDCB induced apoptosis is cell line-dependent.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Bisbenzimidazole; Caspase 3; Caspases; Cell Division; Cell Line; Cell Nucleus; Coprinus; Culture Media; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Female; Fluorescent Dyes; HeLa Cells; Humans; Plant Extracts; Uterine Cervical Neoplasms