cytochrome-c-t and Uterine-Cervical-Neoplasms

As one of the frequent malignancies, breast cancer (BCa) is the foremost reason for cancer-related deaths among women. The role of Human papillomavirus (HPV) in chemoresistance has rarely been investigated in previous studies. The current study sets out to the possible role of HPV in BCa chemoresistance. In this research, 90 BCa tissue and 33 normal breast tissue were collected. We evaluated the presence of the HPV genome along with the viral (E2, E6, E7) and cellular gene expression associated with cell resistance to death. Statically significant differences in the prevalence of HPV between the BCa group (25.2% or 23/90) and the control group (21.8% or 7/32) were not found. HPV-16 and HPV-18 genotypes were the abundant HPV genotypes. Resistance to the Adriamycin-Cyclophosphamide (AC), paclitaxel regimen was elevated in the HPV- group (56/70) in comparison to the HPV+ group (14/70). Nevertheless, there was no significant difference in the prevalence of resistance to AC + paclitaxel + triple-negative breast cancer combination therapy between the HPV+ group (9/20) and in the HPV- group (11/20). In the BCa group in contrast to the control group, the expression level of Bcl-2, BCL-XL, and c-IAP2 demonstrated a significant decrease, while, the expression level of cytochrome C and caspase 3 was significantly increased. This study suggests that HPV infection might contribute to BCa chemoresistance through disrupt cellular genes involved in cell death.

Topics: Breast Neoplasms; Caspase 3; Cytochromes c; Drug Resistance, Neoplasm; Female; Human Papillomavirus Viruses; Humans; Oncogene Proteins, Viral; Paclitaxel; Papillomaviridae; Papillomavirus Infections; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

<b>Background and Objective:</b> Andaliman is a wild plant in Indonesia and it has been used for centuries as traditional medicine. This study aimed to evaluate the effect of methanol extract of andaliman on apoptosis cancer cells via cytochrome c protein. <b>Materials and Methods:</b> The rats are divided into 5 groups. K: Control, K₊: Cancer model rats, P₁: A dose of 100 mg/b.wt./day of andaliman, P₂: A dose of 200 mg/b.wt./day and P₃: A dose of 400 mg/kg/b.wt./day for 30 days. The rats were dissected, then the cervical tissue was prepared on paraffin blocks, given Immunohistochemistry staining with cytochrome c antibody. <b>Results:</b> There was a significant difference in body and cervical weight (p<0.01). The histology also showed a significant difference between each treatment (p<0.01) in cytochrome c. The highest cytochrome c expression was at P₂ and the lowest was at K_-. <b>Conclusion:</b> Andaliman methanol extract can thus be developed into a cervical cancer drug candidate because it can reduce the positive index of cytochrome c in cervical histology.

Topics: Animals; Cytochromes c; Disease Models, Animal; Female; Indonesia; Rats; Uterine Cervical Neoplasms; Zanthoxylum

Splicing factor poly(rC)-binding protein 1 (PCBP1) is a novel tumor suppressor that is downregulated in several cancers thereby regulating tumor formation and metastasis. However, the involvement of PCBP1 in apoptosis of cancer cells and the molecular mechanism remains elusive. On this basis, we sought to investigate the role of splicing factor PCBP1 in the apoptosis in human cervical cancer cells.. To investigate PCBP1 functions in vitro, we overexpressed PCBP1 in human cervical cancer cells. A series of cytological function assays were employed to study to the role of PCBP1 in cell proliferation, cell cycle arrest and apoptosis.. Overexpression of PCBP1 was found to greatly repress proliferation of HeLa cells in a time-dependent manner. It also induced a significant increase in G2/M phase arrest and apoptosis. Furthermore, overexpressed PCBP1 favored the production of long isoforms of p73, thereby inducing upregulated ratio of Bax/Bcl-2, the release of cytochrome c and the expression of caspase-3.. Our results revealed that PCBP1 played a vital role in p73 splicing, cycle arrest and apoptosis induction in human cervical carcinoma cells. Targeting PCBP1 may be a potential therapeutic strategy for cervical cancer therapy.

Topics: Apoptosis; bcl-2-Associated X Protein; Carrier Proteins; Caspase 3; Cell Line, Tumor; Cytochromes c; Female; HeLa Cells; Humans; Protein Isoforms; Proto-Oncogene Proteins c-bcl-2; RNA Splicing Factors; Uterine Cervical Neoplasms

Photodynamic Therapy (PDT) is a photoactivation or photosensitization process, wherein vitamin K3 (Vit K3) serves as a photosensitizer to produce Reactive Oxygen Species (ROS) against bacteria at appropriate wavelengths. In this study, we used Vit K3 treatment combined with Ultraviolet radiation A (UVA) to produce photodynamic effects on cervical cancer.. The dose-concentration relationship between Vit K3 treatment and UVA on tumor cells was analyzed through the Cell Counting Kit-8 method. Then, the morphological characteristics of apoptosis cells were observed through fluorescent staining and fluorescence microscopy. Apoptosis after treatment with Vit K3 treatment, UVA, and Vit K3 treatment plus UVA was further observed through Western blot analysis, flow cytometry, and TUNEL assay. The xenograft models from HeLa cells were established for the exploration of the photodynamic effect of Vit K3 treatment on cervical cancer in vivo.. Vit K3 treatment plus UVA reduced tumor cell viability in a dose-dependent manner. Further studies indicated that Vit K3 treatment plus UVA can inhibit tumor growth and enhance the apoptosis of cervical cancer cells. In the combination group, the expression levels of cleaved caspase-3, cleaved caspase-9, B-cell lymphoma- extra large (Bcl-xl), and cytochrome c (cyt-c) increased obviously, whereas the expression level of Bcell lymphoma 2 (Bcl-2) decreased relative to the expression levels of UVA- or Vit K3-treated cells. In the in vivo experiments, tumor growth was inhibited significantly in the VitK3 treatment plus UVA group. Additionally, we demonstrated that the combination therapy mediated an increase in cleaved caspase-3 and cleaved caspase-9 expression and decrease in Bcl-2 expression in vivo.. Our results showed that Vit K3 treatment combined with UVA exerted photodynamic effects on cervical cancer cells by activating mitochondrial apoptosis pathways.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Caspase 3; Caspase 9; Combined Modality Therapy; Cytochromes c; Drug Discovery; Female; Gene Expression Regulation; HeLa Cells; Humans; Mitochondria; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; Signal Transduction; Ultraviolet Rays; Uterine Cervical Neoplasms; Vitamin K 3

Newcastle disease virus (NDV) is a potential oncolytic virus for the cancer treatment due to its ability to replicate in tumor cells. The aim of this study was to evaluate the in vitro anticancer properties of Hitchner B1 (HB1) strain of NDV on TC-1 cell line and underlying molecular mechanisms. The cytolytic effects of oncolytic HB1 strain of NDV was determined by lactate dehydrogenase (LDH) release assay. Apoptosis, intracellular reactive oxygen species (ROS) levels, cleaved caspase-3 and autophagy were evaluated by flow cytometry. Cytochrome-C and survivin protein levels were distinguished by Enzyme-Linked Immunosorbent Assay (ELISA). Our results from LDH method showed that the viability of the TC-1 cell line following HB1 NDV infection was dose-dependent and decreased significantly with increasing the dose of HB1 NDV infection (MOIs: 5, 10, and 15). Other evaluations also revealed that HB1 strain of NDV potentially led to the ROS production, and apoptosis and autophagy induction in TC-1 cell line in a dose-dependent manner. The in vitro experiments also presented that NDV treatment significantly up-regulated the expression of cytochrome-C and down-regulated the expression of survivin, as detected by ELISA assay. Our results confirmed that the HB1 NDV could be introduced as a powerful candidate for the therapy of cervical cancer. However, further examinations are needed to explain the underlying mechanisms of the HB1 NDV against TC-1 cell line and cervical cancer.

Topics: Animals; Apoptosis; Autophagy; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Female; Humans; Newcastle Disease; Newcastle disease virus; Oncolytic Virotherapy; Uterine Cervical Neoplasms

Human papillomaviruses (HPVs), such as HPV 16 and HPV 18 are related to cervical cancer. Therefore, it is important to inhibit HPV-positive cervical cancer for treating cervical cancer. This study is aiming at investigating the proposed molecular mechanism, which underlies the antineoplastic potential of the aqueous extract of juglone of HPV-positive cervical cancer cells. According to the results, it is showed that, juglone prohibited HPV positive cervical cancer cells' growth through dose-dependent way. Nevertheless, when pin 1 was knocked down, the proliferation inhibition reduced. The detection of apoptosis and cell cycle also illustrated that juglone influenced HPV positive cells. Western blot expressed the influence mechanism that it affected the B-cell lymphoma 2 (Bcl-2) family and later activated the Caspase-depended apoptosis way. It is contributable for this study to understand the mechanism of inhibiting HPV positive cells by juglone and it also provides an effective strategy for the application of it in the future.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Female; Gene Expression Regulation; Human papillomavirus 16; Human papillomavirus 18; Humans; Mitochondria; Naphthoquinones; Up-Regulation; Uterine Cervical Neoplasms

Cervical cancer is the fourth most lethal human malignancy and the leading cause of death among females around the world. Many antitumor agents have microbial origins. 5'-epi-SPA-6952A is a new 24-membered macrolide isolated from the cultured broth of Streptomyces diastatochromogenes. Therefore, we studied the activity and molecular mechanism of 5'-epi-SPA-6952A in human cervical carcinoma HeLa cell. The results showed that 5'-epi-SPA-6952A significantly inhibited cell proliferation and migration. In addition, 5'-epi-SPA-6952A obviously increased the production of intracellular reactive oxygen species and DNA damage in HeLa cells. Moreover, nuclear shrinkage of cells, decrease in mitochondrial membrane potential, and upregulation of Bax/Bcl-2 ratio resulted in the release of cytochrome c, and activation of caspase-9/3 was observed in HeLa cells treated with 5'-epi-SPA-6952A, which means it enhanced the intrinsic mitochondrial apoptosis. Besides, DNA-damage associated proteins poly (ADP-ribose) polymerase (PARP) and p53 were also studied, and the expressions of cleaved-PARP and p53 were drastically increased in HeLa cells treated with 5'-epi-SPA-6952A. Furthermore, we confirmed that 5'-epi-SPA-6952A affected the survival of HeLa cells by blocking cell cycle progression in the G1 phase. Taken together, the results shows that 5'-epi-SPA-6952A significantly inhibited HeLa cells proliferation via intrinsic mitochondrial apoptosis, cell cycle arrest, and blocking cell migration.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cytochromes c; DNA Damage; Female; G1 Phase Cell Cycle Checkpoints; HeLa Cells; Humans; Macrolides; Membrane Potential, Mitochondrial; Mitochondria; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Streptomyces; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Cervical carcinoma continues to be one of the most dangerous cancer types, and more effective therapies are urgently needed for cervical carcinoma treatment. Mitochondria-associated Mitofusin 2 has influence on the progression of many cancers. In the current study, we aimed to focus on the cell apoptotic effects of Mfn2 on cervical carcinoma HeLa cells in vitro and to try to explore its underlying mechanisms. Moreover, we investigated the anticancer potential of Mfn2 in a cervical carcinoma mouse model.. Adenovirus-Mfn2 (Adv-Mfn2) was used to deliver mfn2 into HeLa cells and tumour tissues in a nude mouse model. CCK-8, TUNEL assay, Western blot and immunohistochemical staining were performed to detect the effects of Mfn2. The mRNA level of Mfn2 was determined by quantitative realtime PCR (qRT-PCR) analysis. The effect of Mfn2 on cell apoptosis was investigated by flow cytometry. Flow cytometry was used to assess the change of the mitochondrial membrane potential of the cells treated with JC-1 assay. Mfn2, Bax, Bcl-2, cytochrome c, cleaved caspase-3, and cleaved caspase-9 protein levels were analysed by Western blot.. Data from CCK-8 and flow cytometry showed that Mfn2 could inhibit proliferation and induce apoptosis in a dose- and time-dependent manner in HeLa cells. JC-1 test results revealed that the membrane potential of the mitochondrial decreased in a dose-dependent manner in HeLa cells after Adv-Mfn2 treatment. The data from Western blot confirmed that higher cytosolic amounts of cytochrome c with increasing doses of Adv-Mfn2 signified the onset of the intrinsic apoptotic pathway. Levels of cleaved caspase-3 and cleaved caspase-9 increased in HeLa cells with Adv-Mfn2 treatment. We also found significant increases in the Bax level and a decreased Bcl-2 level with Adv-Mfn2 treatment. We further confirmed that Mfn2 could significantly inhibit the growth of the cervical tumour in the xenografted cervical carcinoma mouse model. After a 9-day-treatment, the tumours of the Adv-mfn2 group were inhibited and induced into apoptosis. The results demonstrated that the overexpression of Mfn2 could not only increase the levels of Bax and Bid in cervical tumour cells but also decrease the phosphorylation of Bad and the expression of Bcl-2.. These studies suggested that the overexpression of Mfn2 could trigger cervical tumour apoptosis in vitro and in vivo, which was related to the mitochondrial pathway, and may provide a new treatment target for cervical carcinoma.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cytochromes c; Female; GTP Phosphohydrolases; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Mitochondrial Proteins; Plasmids; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Transplantation, Heterologous; Uterine Cervical Neoplasms

A dopamine receptor antagonist, Thioridazine (TDZ) is known for its cytotoxic activity against various cancers and its role in combinational chemotherapy is being actively investigated. Several molecular targets of TDZ have been studied to delineate its anticancer activities, with contrasting findings in different cancer types. Moreover, the underlying mechanism of cell death from TDZ treatment is not well defined. In the current study, we studied TDZ mediated cell death mechanism employing cervical cancer cells. TDZ treatment induced nuclear condensation, mitochondrial membrane potential loss, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3 substantiating mitochondrial pathways of apoptosis in cells. TDZ induced ROS generation and up-regulation of ER stress linked proteins, such as CHOP, BiP etc. ER stress and apoptosis caused by TDZ were prevented by ROS inhibitor N-acetyl-L-cysteine (NAC) and protein synthesis inhibitor cycloheximide. In TDZ mediated cytocidal cellular process, autophagy acted as a cell survival factor as the inhibition of autophagy by 3-Methyladenine resulted in increased cell death. TDZ induced apoptosis was associated with decreased Bcl-2 expression and the overexpression of Bcl-2 resulted in inhibition of apoptosis. Studies in Bax-Bak knock-out cell model indicated that TDZ trigger both the Bax-Bak dependent and independent apoptosis through ROS. In the presence of Bax and Bak, cells are more sensitised to death than in the absence of these proteins. Both Bax-Bak dependent and independent apoptosis were significantly inhibited by ROS inhibitor NAC. Conclusively, TDZ induced Bax-Bak dependent and independent apoptosis by enhancing ROS production followed by ER stress.

Topics: Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Autophagy; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Caspase 3; Cytochromes c; Dose-Response Relationship, Drug; Endoplasmic Reticulum Stress; Female; Fibroblasts; HeLa Cells; Humans; Mice; Mitochondria; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Thioridazine; Time Factors; Uterine Cervical Neoplasms

Myostatin (Mstn) is postulated to be a key determinant of muscle loss and cachexia in cancer. However, no experimental evidence supports a role for Mstn in cancer, particularly in regulating the survival and growth of cancer cells. In this study, we showed that the expression of Mstn was significantly increased in different tumor tissues and human cancer cells. Mstn knockdown inhibited the proliferation of cancer cells. A knockout (KO) of Mstn created by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 (CRISPR/Cas9) induced mitochondria-dependent apoptosis in HeLa cells. Furthermore, KO of Mstn reduced the lipid content. Molecular analyses demonstrated that the expression levels of fatty acid oxidation-related genes were upregulated and then increased rate of fatty acid oxidation. Mstn deficiency-induced apoptosis took place along with generation of reactive oxygen species (ROS) and elevated fatty acid oxidation, which may play a role in triggering mitochondrial membrane depolarization, the release of cytochrome c (Cyt-c), and caspase activation. Importantly, apoptosis induced by Mstn KO was partially rescued by antioxidants and etomoxir, thereby suggesting that the increased level of ROS was functionally involved in mediating apoptosis. Overall, our findings demonstrate a novel function of Mstn in regulating mitochondrial metabolism and apoptosis within cancer cells. Hence, inhibiting the production and function of Mstn may be an effective therapeutic intervention during cancer progression and muscle loss in cachexia.

Topics: A549 Cells; Animals; Antioxidants; Apoptosis; Cachexia; Caspases; Cell Line, Tumor; Cell Proliferation; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Cas Systems; Cytochromes c; Epoxy Compounds; Fatty Acids; Female; Gene Knockout Techniques; HEK293 Cells; HeLa Cells; Humans; Lipid Metabolism; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Myostatin; Oxidation-Reduction; Reactive Oxygen Species; Uterine Cervical Neoplasms; Xenograft Model Antitumor Assays

Cancer cells down-regulate different genes to give them a selective advantage in invasiveness and/or metastasis. The SLC25A26 gene encodes the mitochondrial carrier that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix, required for mitochondrial methylation processes, and is down-regulated in cervical cancer cells. In this study we show that SLC25A26 is down-regulated due to gene promoter hypermethylation, as a mechanism to promote cell survival and proliferation. Furthermore, overexpression of SLC25A26 in CaSki cells increases mitochondrial SAM availability and promotes hypermethylation of mitochondrial DNA, leading to decreased expression of key respiratory complex subunits, reduction of mitochondrial ATP and release of cytochrome c. In addition, increased SAM transport into mitochondria leads to impairment of the methionine cycle with accumulation of homocysteine at the expense of glutathione, which is strongly reduced. All these events concur to arrest the cell cycle in the S phase, induce apoptosis and enhance chemosensitivity of SAM carrier-overexpressing CaSki cells to cisplatin.

Topics: Adenosine Triphosphate; Amino Acid Transport Systems; Apoptosis; Calcium-Binding Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cytochromes c; DNA Methylation; DNA, Mitochondrial; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Methionine; Mitochondria; Promoter Regions, Genetic; S-Adenosylmethionine; Uterine Cervical Neoplasms

To investigate the mechanism underlying the inhibitory effect of STOML-2 overexpression on apoptosis of human cervical squamous carcinoma Siha cells.. Siha cells were transfected with an adenoviral vector carrying STOML-2, and 72 h later STOML-2 expression and the proliferation of the cells were detected by Western blotting and MTT assay. The transfected cells were treated with IC50 Cisplatin for 24 h, and the morphological changes of cells were observed using fluorescence, and the cell apoptosis was analyzed using flow cytomerty; the expression levels of proteins related with mitochondrial apoptosis pathway, including caspase-3, cleaved caspase-3, Bcl-2, Bax and cytochrome C (Cyt C), were detected by Western blotting.. Western blotting showed a significantly increased STOML-2 expression in the transfected cells. Overexpression of STOML-2 obviously promoted the proliferation of Siha cells. The STOML-2-overexpressing cells exhibited an obvious resistance to IC50 Cisplatin-induced apoptosis as shown by both fluorescence microscopy and flow cytometry and presented with decreased expressions of cleaved caspase-3, Bax, and cytosol Cyt C and increased expressions of caspase-3, Bcl-2, and mitochondrial Cyt C.. Overexpression of STOML-2 can enhance the proliferation of Siha cells by inhibiting cell apoptosis possibly through the mitochondrial apoptosis pathway.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Blood Proteins; Carcinoma, Squamous Cell; Caspase 3; Cell Line, Tumor; Cisplatin; Cytochromes c; Female; Flow Cytometry; Humans; Membrane Proteins; Mitochondria; Uterine Cervical Neoplasms

MCPH1, initially identified as an hTERT repressor, has recently been implicated in mediating DNA damage response and maintaining chromosome integrity. This study is to investigate its potential role in the onset of cervical cancer. In the study, decreased expression of MCPH1 was observed in 19 of 31 cases (61.3%) at mRNA level and 44 of 63 cases (69.8%) at protein level of cervical tumor tissues compared with the paired nontumor tissues. Reduced MCPH1 protein expression was significantly associated with high-tumor grade (1 vs. 3 P = 0.013; 2 vs. 3 P = 0.047). In addition to inhibit SiHa cell migration and invasion, the overexpression of MCPH1 inhibited cervical cancer cells growth through inducing S phase arrest and mitochondrial apoptosis. Further analysis demonstrated cyclinA2/CDK2, CDC25C-cyclinB/CDC2, and p53/p21 pathways were involved in the MCPH1 overexpression-induced S phase arrest. Moreover, the overexpression of MCPH1 activated mitochondrial apoptosis through regulating several apoptosis-related proteins such as p53, Bcl-2, Bax, cytochrome c, caspase-3, and PARP-1. Our findings indicate that downregulated MCPH1 correlates with tumor progression in cervical cancer, and MCPH1 has an important role in regulating cell growth through regulating the cell cycle and apoptosis. Thus, it may be a crucial tumor suppressor gene and a novel candidate therapeutic target for cervical cancer.

Topics: Base Sequence; Caspase 3; Cell Cycle Proteins; Cell Division; Cell Line; Cytochromes c; Cytoskeletal Proteins; DNA Primers; Female; Humans; Nerve Tissue Proteins; Real-Time Polymerase Chain Reaction; Signal Transduction; Uterine Cervical Neoplasms

Coumarins and chalcones are secondary plant metabolites which have shown an array of pharmacological properties including anti-tumour activity. We have previously reported on the synthesis and anti-proliferative activity of a series of novel coumarin-chalcone hybrids. Now we report on the in vivo efficacy as well as mechanism of action of the most potent molecule of the series, S009-131. Oral administration of this molecule resulted in regression of tumours induced by HeLa cell xenografts in nod SCID mice. The molecule inhibited proliferation of cervical cancer cells (HeLa and C33A) by inducing apoptosis and arresting cell cycle at G2/M phase. Apoptosis was induced through induction of caspase-dependent intrinsic pathway and alterations in the cellular levels of Bcl-2 family proteins. The mitochondrial transmembrane potential got highly depleted in S009-131 treated cells due to an increase in Bax/Bcl-2 ratio and intracellular ROS. The molecule induced release of cytochrome c into the cytosol and activation of initiator caspase-9 and executioner caspases-3/7. Tumour suppressor protein p53 and its transcriptional target PUMA were up regulated, suggesting their role in mediating the cell death. These results suggest that S009-131 is a potent candidate for the chemotherapy of cervical carcinoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Proliferation; Chalcone; Chalcones; Coumarins; Cytochromes c; Female; G2 Phase Cell Cycle Checkpoints; Heterografts; Humans; M Phase Cell Cycle Checkpoints; Membrane Potential, Mitochondrial; Mice; Mice, SCID; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Uterine Cervical Neoplasms

Goniothalamus species (Annonaceae) is a shrub that grows in the rainforest of tropical Asia. Several compounds have been isolated and exhibit the potential use for cancer treatment. In this work, altholactone isolated from Goniothalamus macrophyllus was investigated for its cytotoxicity, apoptosis signalling and the expression of cancer-related genes in the cervical carcinoma HeLa cells. Cytotoxicity was evaluated by MTT assay. Apoptotic characteristics were evaluated by morphological studies. Caspase-3 activity was detected using a fluorogenic substrate. Cytochrome c release from mitochondria and protein Bid were determined by Western blotting and cancer-related genes expression by RT-PCR. The results demonstrated that altholactone was cytotoxic to HeLa (IC50  = 9.6 μg/mL), and apoptotic cell death was manifested by appearance of chromatin condensation and caspase-3 activation, which was inhibited by specific inhibitors of both caspase-8 and -9. Release into the cytosol of cytochrome and cleavage of Bid occurred. Altholactone also caused a decrease in bcl-2 and an increase in p53 expression. These unique properties of altholactone suggest a potential for cancer chemotherapy.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Caspase 8; Caspase 9; Cytochromes c; Female; Furans; Gene Expression Regulation, Neoplastic; Goniothalamus; HeLa Cells; Humans; Proto-Oncogene Proteins c-bcl-2; Pyrones; Signal Transduction; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Small aromatic compounds like flavonoids can intercalate with DNA molecules bringing about conformational changes leading to reduced replication and transcription. Here, we have examined one dietary flavonoid, quercetin (found in many fruit and vegetables), for possible anti-cancer effects, on HeLa cells originally derived from a case of human cervical cancer.. By circular dichroism spectroscopy we tested whether quercetin effectively interacted with DNA to bring about conformational changes that would strongly inhibit proliferation and migration of the HeLa cells. Cytotoxic effects of quercetin on cancer/normal cells, if any, were determined by MTT assay and such depolarization of mitochondrial membrane potential, as a consequence of quercetin treatment, and accumulation of reactive oxygen species (ROS) also were studied, by FACS analysis and expression profiles of different anti- and pro-apoptotic genes and their products were determined.. Quercetin intercalated with calf thymus cell DNA and HeLa cell DNA and inhibition of anti-apoptotic AKT and Bcl-2 expression were observed. Levels of mitochondrial cytochrome-c were elevated and depolarization of mitochondrial membrane potential occurred with increase of ROS; upregulation of expression of p53 and caspase-3 activity were also noted. These alterations in signalling proteins and externalization of phosphotidyl serine residues were involved with initiation of apoptosis. Reduced AKT expression suggested reduction in cell proliferation and metastasis potential, with arrest of the cell cycle at G2/M.. Quercetin would have potential for use in cervical cancer chemotherapy.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Movement; Cell Proliferation; Cell Survival; Circular Dichroism; Cytochromes c; DNA; DNA Fragmentation; DNA, Neoplasm; Female; Flow Cytometry; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Genes, bcl-2; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; Membrane Potential, Mitochondrial; Nucleosomes; Quercetin; Reactive Oxygen Species; Signal Transduction; Transcriptome; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

In the present study, a biologically active 4-(trifluoromethyl)phenyl piperazin moiety was linked to a 2,2- dimethyl -2H-benzopyran template to generate (3R,4S)-2,2-dimethyl-6-nitro-4-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl) chroman -3-ol (C110g), and the cellular and molecular mechanisms by which C110g exerts cytotoxic effects on the HeLa human cervical cancer cell line were further investigated. C110g suppressed the viability of HeLa cells in both concentration- and time-dependent manner (IC50 of 17 µM) by inducing DNA damage and G1 cell cycle arrest. Characteristic changes in nuclear morphology and Annexin V/PI staining pointed to apoptosis as the mode of cell death. The levels of p53 and p21 were increased in the C110g-treated cells, with a corresponding increase in Bax/Bcl-2 protein ratio. Subsequently, C110g induced the cytoplasmic release of cytochrome c from the mitochondria accompanied by a decreased mitochondrial membrane potential and activation of caspase-3 and -9. These results confirmed that the C110g transduced the apoptotic signal via the mitochondrial pathway. Caspase-8, typically associated with the initiation of the death receptor pathway, was activated, suggesting the extrinsic pathway might also be involved. However, C110g did not result in reactive oxygen species (ROS) generation. Taken together, these findings indicate that the DNA damage-dependent p53-regulated mitochondrial pathway as well as the extrinsic pathway play a crucial role in C110g-induced apoptosis, which provide a better understanding of the molecular mechanisms of trifluoromethyl benzopyrans in cervical cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Benzopyrans; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Enzyme Activation; Female; G1 Phase Cell Cycle Checkpoints; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Cisplatin remains one of the most effective current chemotherapeutic agents; however, metal complexes synthesis has increased in order to produce new anti-neoplastic drugs with DNA binding and apoptotic activities in tumor cells and less toxicity for patients. In this study, we evaluated the cytotoxic activity of a novel copper(II) complex (LQM402) against cervical cancer cell lines and found that LQM402 exhibited selective cytotoxicity against HeLa and Ca Ski cells. FITC-annexin assay and DNA fragmentation indicated that apoptosis could be involved in HeLa cell death. Caspase 3/7 and cytochrome c analysis by immunoblotting suggest the intrinsic pathway. LQM402 is a lipid peroxidation inductor according to TBARS production. Additionally, the Ames and micronucleus tests demonstrated non-genotoxic activity for this compound in Salmonella typhimurium and CD1 mice, respectively. Therefore, LQM402 may be a promising and safe anti-cervical cancer compound.

Topics: Animals; Annexins; Apoptosis; Brain Chemistry; Caspase 3; Caspase 7; Cell Line, Tumor; Coloring Agents; Coordination Complexes; Copper; Cytochromes c; Cytosol; DNA Fragmentation; Female; Flow Cytometry; Fluorescein-5-isothiocyanate; HeLa Cells; Humans; In Situ Nick-End Labeling; Lipid Peroxidation; Mice; Micronucleus Tests; Mutagenicity Tests; Mutagens; Oxidants; Phosphatidylserines; Rats; Salmonella typhimurium; Tetrazolium Salts; Thiazoles; Uterine Cervical Neoplasms

Antrodia camphorata is a Chinese herb indigenous to Taiwan. Previous reports demonstrated that it could induce apoptosis in some cancer cells. The purpose of this study was to investigate the apoptotic effect of the crude extract of A. camphorata in cervical cancer cells. Two human cervical cancer cell lines, HeLa and C-33A, were treated with extract of A. camphorata (10-1000 μg/mL). We found that A. camphorata extract was cytotoxic to both cervical cancer cells in a dose- and time-dependent manner as examined by MTT assay. Treatment with A. camphorata extract at 400 μg/mL induced a 2.3- and 4.4-fold increase in oligonucleosome formation from the cleaved chromosomal DNA in HeLa and C-33A cells, respectively. A. camphorata extract also activated caspase-3, -8, and -9 activities and increased the cytosolic level of cytochrome c in both cell lines as the dosage increased. Furthermore, A. camphorata extract increased expressions of Bak, Bad and Bim, while decreasing expressions of Bcl-2 and Bcl-xL of the Bcl-2 family proteins in HeLa and C-33A cells. The expression of IAP proteins, XIAP and survivin, was also decreased in both cervical cancer cells after treatment with A. camphorata. Our in vitro study suggests that A. camphorata is cytotoxic to cervical cancer cells through both extrinsic and intrinsic apoptotic mechanisms. It could be used as a novel phytotherapeutic agent or auxiliary therapy in the treatment of cervical cancer.

Topics: Antrodia; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; Bcl-2-Like Protein 11; bcl-Associated Death Protein; Caspases; Chromosomes, Human; Cytochromes c; DNA; Dose-Response Relationship, Drug; Female; Gene Expression; HeLa Cells; Humans; Membrane Proteins; Molecular Targeted Therapy; Nucleosomes; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Flavonoids have antitumoral properties and may be attractive candidates as anticancer therapy. Isoliquiritigenin which is a constituent of licorice (Glycyrrhiza inflata), a plant commonly used in traditional Uyghur medicine in Xinjiang, China, was studied for antiproliferative and apoptotic activity in human cervical cancer cells, Ca Ski, SiHa, HeLa, and C-33A. Its molecular mechanism of action was specifically examined in Ca Ski cells. Isoliquiritigenin decreased cell viability, induced cell accumulation in G2/M and morphological and biochemical features of apoptosis in the four cancer cell lines. In Ca Ski cells, isoliquiritigenin led to a downregulation of HPV16 E6 expression associated with an increase of p53 and p21 levels, enhanced expression of Bax and decreased expression of Bcl-2 and Bid proform triggering dissipation of the mitochondrial membrane potential, released cytochrome c to the cytosol followed by activation of caspase cascade with cleavage of caspase-9, caspase-3, and PARP. Caspase-8 was also cleaved. Moreover treatment with a pan-caspase inhibitor prevented apoptosis. As Ca Ski cells are representative of carcinoma naturally occurring in the cervix, our results suggest a potential benefit of isoliquiritigenin for cervical cancer prevention and treatment.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Caspase Inhibitors; Caspases; Cell Line, Tumor; Chalcones; Cytochromes c; Down-Regulation; Drugs, Chinese Herbal; Female; Glycyrrhiza; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Oncogene Proteins, Viral; Phytotherapy; Proto-Oncogene Proteins c-bcl-2; Repressor Proteins; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Sarsasapogenin is a sapogenin from the Chinese medical herb Anemarrhena asphodeloides Bunge. In the present study, we revealed that sarsasapogenin exhibited antitumor activity by inducing apoptosis in vitro as determined by Hoechst staining analysis and double staining of Annexin V-FITC/PI. In addition, cell cycle arrest in G2/M phase was observed in sarsasapogenin-treated HeLa cells. Moreover, the results revealed that perturbations in the mitochondrial membrane were associated with the deregulation of the Bax/Bcl-2 ratio which led to the upregulation of cytochrome c, followed by activation of caspases. Meanwhile, treatment of sarsasapogenin also activated Unfolded Protein Response (UPR) signaling pathways and these changes were accompanied by increased expression of CHOP. Salubrinal (Sal), a selective inhibitor of endoplasmic reticulum (ER) stress, partially abrogated the sarsasapogenin-related cell death. Furthermore, sarsasapogenin provoked the generation of reactive oxygen species, while the antioxidant N-acetyl cysteine (NAC) effectively blocked the activation of ER stress and apoptosis, suggesting that sarsasapogenin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways. Taken together, the results demonstrate that sarsasapogenin exerts its antitumor activity through both reactive oxygen species (ROS)-mediate mitochondrial dysfunction and ER stress cell death.

Topics: Anemarrhena; Antineoplastic Agents; bcl-2-Associated X Protein; Cell Cycle Checkpoints; Cinnamates; Cytochromes c; Drugs, Chinese Herbal; Endoplasmic Reticulum Stress; Female; G1 Phase Cell Cycle Checkpoints; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; Mitochondria; Mitochondrial Membranes; Reactive Oxygen Species; Spirostans; Thiourea; Transcription Factor CHOP; Unfolded Protein Response; Uterine Cervical Neoplasms

To explore the molecular mechanism of realgar-induced apoptosis of cervical cancer cells.. The cervical cancer cell line Siha was used to determine the cell viability and apoptosis after treatment with realgar using MTT assay and flow cytometry. The activities of caspase-3, -8, and -9 were detected by fluorescence resonance energy transfer technology and colorimetric assay, while the levels of Bcl-2, cytochrome c, and Bax were detected by Western blot method.. Induction of apoptosis by realgar was detected in Siha cell line in a dose-dependent manner. The apoptosis was accompanied by a significant increase in cytochrome c release and activation of caspase-3 and caspase-9 but not caspase-8. Further, the realgar-induced apoptosis was inhibited by a broad-spectrum caspase inhibitor, a caspase-3 inhibitor, and a caspase-9 inhibitor but not by a caspase-8 inhibitor. Bcl-2 and Bax protein expressions were not changed by realgar.. The induction of apoptosis by realgar is mediated through a cytochrome c-dependent pathway, which sequentially activates caspase-9 and caspase-3.

Topics: Apoptosis; Arsenicals; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Female; Fluorescence Resonance Energy Transfer; Humans; Sulfides; Uterine Cervical Neoplasms

Artemisia princeps Pampanini is widely used in Eastern traditional medicine for the treatment of circulatory disorders, such as, dysmenorrhea, hematuria, hemorrhoids, and inflammation, and is also used to treat chronic conditions, such as, cancers, ulcers, and digestive disorders.. The purpose of this study is to investigate the effect of a standardized flavonoid-rich fraction of Artemisia princeps Pampanini cv. Sajabal (FRAP) on the induction of apoptosis and the molecular mechanism involved in human cervical cancer HeLa cells.. Human cervical cancer HeLa cells were treated with FRAP and apoptosis was detected by cell morphologic observation, annexin-V-PI staning and western blot analysis on the expression of protein associated with cell death.. FRAP led to the cleavages of caspase-3, -8, and -9 and the cleavage of poly (ADP-ribose) polymerase (PARP) in HeLa cells. Caspase-3 inhibitor (z-DEVD-fmk), caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD), and broad caspase inhibitor (z-VAD-fmk) significantly suppressed the FRAP-induced accumulation of annexin V positive cells. Furthermore, it was found that FRAP caused a loss of mitochondrial membrane potential (MMP) and the release of cytochrome c to the cytosol. Furthermore, the overexpression of Bcl-xL significantly prevented FRAP-induced apoptosis, MMP changes, and the activations of caspase-3, -8, and -9. Interestingly, pretreatment with caspase-8 inhibitor significantly reduced the FRAP-induced activation of caspase-3 but not that of caspase-9, whereas the caspase-3 inhibitor, z-DEVD-fmk, markedly attenuated the FRAP-induced activation of caspase-8. In BALB/c(nu/nu) mice bearing a HeLa xenograft, FRAP dosed at 25 or 50mg/kg significantly inhibited tumor growth.. Our results indicate caspase-mediated activation of the mitochondrial death pathway plays a critical role in the FRAP-induced apoptosis of HeLa cells and that FRAP inhibits the in vivo tumor growth of HeLa xenograft mice.

Topics: Animals; Apoptosis; Artemisia; bcl-X Protein; Blotting, Western; Caspase Inhibitors; Caspases; Chemical Fractionation; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Female; Flavonoids; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Phytotherapy; Plant Components, Aerial; Plant Extracts; Plants, Medicinal; Poly(ADP-ribose) Polymerases; Time Factors; Transfection; Uterine Cervical Neoplasms; Xenograft Model Antitumor Assays

To investigate the role of Beclin 1 expression on the cisplatin-induced apoptosis in cervical cancer CaSki cells and to explore the potential mechanism underlying this effect.. After overexpression or partial silencing of Beclin 1 in cervical cancer CaSki cells, the transfected group and the control group were treated with cisplatin for 24 hours. The percentage of apoptotic cells were assessed by flow cytometry. The mitochondrial membrane potential and activities of caspase-8/9/3 were detected by JC-1 fluorescence staining and colorimetry. The expression of cytochrome c was measured using a Western blot. The messenger RNA expression of Bax and Bcl-2 were detected by real-time quantitative reverse transcription polymerase chain reaction.. Expression of Beclin 1 protein was up-regulated in overexpressed transfectants of CaSki cells. After treatment with cisplatin, the Beclin 1 overexpression group led to the decrease of mitochondrial membrane potential and increase of activities of caspase-9 and caspase-3, and showed a greater increase in apoptosis than did the nontransfected group. Furthermore, Beclin 1 overexpression resulted in increased cytoplasmic cytochrome c and Bax expression and decreased mitochondrial cytochrome c and Bcl-2 expression.. Overexpression of Beclin 1 in CaSki cells may influence cisplatin-induced apoptosis by mitochondrial dependent pathway.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Beclin-1; Blotting, Western; Caspase 3; Caspase 9; Cisplatin; Cytochromes c; Female; Flow Cytometry; Humans; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Uterine Cervical Neoplasms

In this work, the effects of a pair of positional isomer of ganoderic acids (GAs), namely ganoderic acid Mf (GA-Mf) and ganoderic acid S (GA-S) purified from the fermented mycelia of Ganoderma lucidum, on induction of cell apoptosis and the apoptotic pathway in HeLa cells were investigated. The results demonstrate that both isomers decreased cell population growth on various human carcinoma cell lines by MTT assay, while GA-Mf had better selectivity between normal and cancer cells. The flow cytometry analysis indicated that treatment of HeLa cells with GA-S caused cell cycle arrest in the S phase, while GA-Mf caused cell cycle arrest in the G1 phase. Compared with GA-S, GA-Mf had more potent increase in the number of early and late apoptotic cells. Treatment of HeLa cells with each isomer decreased the mitochondria membrane potential and caused the release of cytochrome c from mitochondria into the cytosol. In addition, stimulation of caspase-3 and caspase-9 activity was observed. The Bax/Bcl-2 ratio was also increased in GA-treated HeLa cells. The results demonstrated that both isomers GA-Mf and GA-S induced apoptosis of human HeLa cells through a mitochondria mediated pathway, but they had the different cell cycle arrest specificity. The findings will be helpful to the development of useful cancer chemopreventive compounds from G. lucidum.

Topics: Apoptosis; Carcinoma; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Line; Cytochromes c; Dose-Response Relationship, Drug; Female; G1 Phase; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mycelium; Reishi; S Phase; Triterpenes; Uterine Cervical Neoplasms

Latex of Euphorbia antiquorum (EA) has inhibitory effects on several different cancer cell lines. However, the molecular mechanism of EA inhibitory effects on human cervical cancer HeLa cell growth has not been explored. EA induced apoptosis, which was characterized by morphological change, DNA fragmentation, increased sub-G1 population, and alterations in levels of apoptosis-associated proteins. Treatment with EA increased cell death and expression levels of caspase-8, -9, and -3. EA suppressed expression of Bcl-2, increased Bax, and reduced cleavage of Bid and the translocation of tBid to the mitochondria and the release of cytochrome c from mitochondria. EA caused a loss of mitochondrial membrane potential (ΔΨm) and an increase in cellular reactive oxygen species (ROS). EA-induced ROS formation was suppressed by cyclosporine A (an inhibitor of the ΔΨm) or allopurinol (an effective scavenger of ROS). EA also increased expression of Fas, FasL, and c-Jun N-terminal kinase (JNK), p38, and mitogen-activated protein kinase (MAPK) and decreased expression of extracellular signal-regulated kinase (ERK) 1/2-p. Co-treatment with the JNK inhibitor SP600125 inhibited EA-induced apoptosis and the activation of caspase-8, -9, and -3. Results of this study provide support for the hypothesis that EA causes cell death via apoptotic pathways in human cervical adenocarcinoma HeLa cells.

Topics: Allopurinol; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 8; Caspase 9; Cyclosporine; Cytochromes c; Euphorbia; Female; HeLa Cells; Humans; JNK Mitogen-Activated Protein Kinases; Latex; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; Uterine Cervical Neoplasms

NF-kappaB activation is known to reduce the efficiency of chemotherapy in cancer treatment. Ursolic acid, a minimally toxic compound, has shown the capability to inhibit NF-kappaB activation in living cells. Here, for the first time, we investigated the effects and mechanisms of NF-kappaB inhibition by ursolic acid on chemotherapy treatment (Taxol or cisplatin) of cancer. ASTC-a-1 (human lung adenocarcinoma), Hela (human cervical cancer) cells, primary normal mouse cells of lung and liver and mouse in vivo model were used. Activity of signal factors (NF-kappaB, Akt, Fas/FasL, BID, Bcl-2, cytochrome c and caspase-8, 3) was used to analyze the mechanisms of ursolic acid-chemo treatment. Ursolic acid-mediated suppression of NF-kappaB drastically reduced the required dosage of the chemotherapeutic agents to achieve identical biological endpoints and enhanced the chemotherapeutic agent-induced cancer cells apoptosis. Chemosensitization by ursolic acid in cancer cells was dependent on the amplified activation of intrinsic pathway (caspase-8-BID-mitochondria-cytochrome c-caspase-3) by augmentation of BID cleavage and activation of Fas/FasL-caspase-8 pathway. Prolonged treatment with relatively low doses of ursolic acid also sensitized cancer cells to the chemotherapeutic agents through suppression of NF-kappaB. Chemosensitization by ursolic acid was observed only in cancer cells, but not in primary normal cells. The inhibitive effect of ursolic acid on NF-kappaB was reversible, and the reversal was not accompanied by a loss in cells viability. By supplementing chemotherapy with minimally toxic ursolic acid, it is possible to improve the efficacy of cancer treatment by significantly reducing the necessary drug dose without sacrificing the treatment results.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Caspase 8; Cell Proliferation; Cells, Cultured; Cisplatin; Cytochromes c; Drug Synergism; Fas Ligand Protein; fas Receptor; Female; Flow Cytometry; Humans; Liver; Luciferases; Lung; Lung Neoplasms; Mice; NF-kappa B; Protein Transport; RNA, Small Interfering; Signal Transduction; Triterpenes; Ursolic Acid; Uterine Cervical Neoplasms

A novel polysaccharide isolated from Angelica sinensis, named APS-1d showed cytotoxic activity towards several cancer cell lines in vitro. However, the precise antitumor mechanisms of this compound are unknown. In this study, we investigated the pro-apoptotic effects of APS-1d in human cervical cancer HeLa cells both in vitro and in vivo, and further elucidated the mechanisms of this action. Inhibition of HeLa cell proliferation was determined by MTT assay and the therapeutic efficacy of APS-1d was evaluated by human cancer xenografts in nude mice. Cell apoptosis was examined with flow cytometry and TUNEL assay. The mechanism of action of APS-1d was investigated by Western blot analysis. APS-1d decreased HeLa cell proliferation in a concentration- and time-dependent manner in vitro. In addition, APS-1d significantly inhibited tumor growth in athymic nude mice. Characteristic manifestations of apoptosis including apoptotic morphological features and the sub- G(0)/G(1) peaks were observed when the cells were treated with APS-1d. Further analysis showed that APS-1d-induced apoptosis was associated with the regulation of Bcl-2 family protein expression, a decrease in the mitochondrial membrane potential, and an increase in the cytosolic cytochrome c levels. Sequentially, APS-1d increased the activities of caspase-9, -3, and poly (ADP-ribose) polymerase in a concentration-dependent manner, however, no obvious activation of Bid and caspase-8 was observed. Pretreatment with Z-LEHD-FMK, a specific inhibitor of caspase-9, significantly attenuated APS-1d-induced cell apoptosis, and activation of caspase-3. Taken together, our studies indicate that APS-1d is capable of inhibiting HeLa cell proliferation and inducing apoptosis in these cells which primarily involves the activation of the intrinsic mitochondrial pathway.

Topics: Angelica sinensis; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Female; Flow Cytometry; HeLa Cells; Humans; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Oligopeptides; Phytotherapy; Plant Extracts; Poly(ADP-ribose) Polymerases; Polysaccharides; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Uterine Cervical Neoplasms; Xenograft Model Antitumor Assays

Aesculetin (1) is an important coumarin found in various plant materials. It has been shown to have antiproliferative effects on several types of human cancer cells, but its effect on cervical cancer cells in vitro is unknown. In this study, we investigated that the cytotoxic effect of 1 on a non-cancer cell line (293) was smaller than on a tumor cell line (HeLa). This is the first report showing the possible mechanism of antiproliferative effect of 1 for the prevention of cervical cancer in cell culture models. It was found that 1 inhibited cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies, generation of reactive oxygen species (ROS), and the accumulation of cells in the sub-G1 phase. Treatment with compound 1 decreased the cell growth in a dose-dependent manner with an IC(50) value of 37.8 microM. Aesculetin-induced apoptosis was correlated with mitochondrial dysfunction (DeltaPsi(m)), leading to the release of cytochrome c from the mitochondria to the cytosol, as well as the proteolytic activation of caspases in HeLa cells. These results indicate that 1 induces apoptosis in HeLa cells through a ROS-mediated mitochondrial dysfunction pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Bisbenzimidazole; Caspases; Cytochromes c; Drug Screening Assays, Antitumor; Female; HeLa Cells; Humans; Mitochondria; Models, Biological; Molecular Structure; Plants, Medicinal; Tumor Cells, Cultured; Umbelliferones; Uterine Cervical Neoplasms

beta-Elemene, a natural compound extracted from over 50 different Chinese medicinal herbs and plants, has been effective in the treatment of hyperplastic and proliferative disorders such as prostatic hypertrophy, hysteromyoma and neoplasms. Our previous studies have demonstrated that beta-elemene exhibits strong inhibitory activity in ovarian cancer cells. The aim of the present study was to assess the effect of beta-elemene on prostate cancer cells as well as other types of tumour cells and to determine whether the effect of beta-elemene on prostate cancer cell death was mediated through the induction of apoptosis.. The MTT assay was used to evaluate the ability of beta-elemene to inhibit cellular proliferation in cancer cells. Cellular apoptosis was assessed by annexin V binding, TUNEL and ELISA-based assays. Caspase activity was measured using a caspases assay kit. The protein levels of Bcl-2, caspases, cytochrome c and poly(ADP-ribose) polymerase (PARP) were analysed by Western blotting.. Here, we showed that beta-elemene had an antiproliferative effect on androgen-insensitive prostate carcinoma DU145 and PC-3 cells. Treatment with beta-elemene also inhibited the growth of brain, breast, cervical, colon and lung carcinoma cells. The effect of beta-elemene on cancer cells was dose dependent, with IC50 values ranging from 47 to 95 microg/ml (230-465 microm). TUNEL assay and flow cytometric analysis using annxin V/propidium iodide staining revealed that the percentage of apoptotic prostate cancer cells was increased by beta-elemene in a dose- and time-dependent manner. Moreover, beta-elemene exposure resulted in a decreased Bcl-2 protein level, increased cytochrome c release, and activated PARP and caspase-3, -7, -9, and -10 in prostate cancer cells.. Overall, these findings suggest that beta-elemene exerts broad-spectrum antitumour activity against many types of solid carcinoma and supports a proposal of beta-elemene as a new potentially therapeutic drug for castration-resistant prostate cancer and other solid tumours.

Topics: Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Humans; In Situ Nick-End Labeling; Inhibitory Concentration 50; Lung Neoplasms; Male; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sesquiterpenes; Time Factors; Uterine Cervical Neoplasms

CMTM5 (CKLF-like MARVEL transmembrane domain-containing member 5) exhibits tumor inhibition activity with frequent epigenetic inactivation in various tumor cell lines including cervical carcinoma (CC) cells. In this paper, we examined the function of CMTM5-v1 (the primary RNA splicing form) in both HeLa and SiHa cells. Overexpression of CMTM5-v1 in both cells can induce apoptosis, but the effects are more obvious in SiHa than that in HeLa. In SiHa cells, restoration of CMTM5-v1 caused disruption of mitochondrial transmembrane potential, release of cytochrome c, activation of caspase3 and cleavage of PARP. General caspase inhibitor almost prevented apoptosis of SiHa cells, suggesting that CMTM5-v1 induces apoptosis mainly through caspase-dependent pathway. These findings verify that CMTM5-v1 inhibits the growth of CC cell lines via inducing apoptosis.

Topics: Apoptosis; Carcinoma; Caspase 3; Cell Proliferation; Chemokines; Cytochromes c; Female; HeLa Cells; Humans; MARVEL Domain-Containing Proteins; Tumor Suppressor Proteins; Uterine Cervical Neoplasms

Cathepsins have long been considered as housekeeping molecules. However, specific functions have also been attributed to each of these lysosomal proteases. Squamous cell carcinoma antigen (SCCA) 1, widely expressed in various uterine cervical cells, is an endogenous cathepsin (cat) L inhibitor. In this study, we investigated whether the cat L-SCCA 1 lysosomal pathway and autophagy were involved in resveratrol (RSV)-induced cytotoxicity in cervical cancer cells. RSV induced GFP-LC3 aggregation as well as increased the presence of LC3-II and autophagosomes as was revealed by electron microscopy in cervical cancer cells. Prolonged treatment of RSV induced cytosolic translocation of cytochrome c, caspase 3 activation and apoptotic cell death. This apoptotic effect was abrogated by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cat B and L, but not by pepstatin A, an inhibitor of cat D. As cervical cancer cells express little cat B, we further studied the role of cat L. RSV induced dissipation of the lysosomal membrane permeability (LMP), leakage and increased cytosolic expression and activity of cat L. Inhibition of cat L by small interference RNA (siRNA) protected cells from RSV-induced cytotoxicity. In contrast, inhibition of SCCA 1 by siRNA promoted RSV-induced cytotoxicity. Inhibition of autophagic response by wortmannin (WT) or asparagine (ASP) resulted in decreased early LC3-II formation, reduced LMP, and abolishment of the increase in RSV-induced cell death. In conclusion, we have identified a new cytotoxic mechanism in which the lysosomal enzyme cat L acts as a death signal integrator in cervical cancer cells. Furthermore, SCCA 1 may play an antiapoptotic role through anti-cat L activity.

Topics: Apoptosis; Autophagy; Caspase 3; Cathepsin L; Cathepsins; Cell Line, Tumor; Cell Proliferation; Cysteine Endopeptidases; Cytochromes c; Cytosol; Enzyme Activation; Extracellular Space; Female; Humans; Intracellular Membranes; Iron; Lysosomes; Models, Biological; Permeability; Protein Transport; Resveratrol; Stilbenes; Uterine Cervical Neoplasms

Cervical cancer is the most common cancer in Indian females and is associated with infection with high risk Human papillomaviruses (HPVs). Curcumin (Diferuloyl methane), a chemopreventive agent, is a natural compound extracted from Curcuma longa that allows suppression of carcinogenesis. The objective of this study was to identify the molecular mechanism of curcumin induced apoptosis in HPV positive cervical cancer HeLa, SiHa and Ca Ski cells. Curcumin causes distinct inhibition of human telomerase reverse transcriptase (hTERT) the catalytic core of telomerase thereby reducing proliferation of cancer cells. Curcumin mediated apoptosis in these cells appears to be due to upregulation of proapoptotic Bax, AIF, release of cytochrome c and down regulation of antiapoptotic Bcl-2, Bcl-XL in HeLa and SiHa. This was accompanied by an increase in caspase-3 and -9 activity, suggesting the role of mitochondria in curcumin mediated apoptotic cell death. Curcumin acts as an anti-inflammatory and anti-proliferative agent by causing down regulation of COX-2, iNOS and cyclin D1 in all the three cell lines but to different extent.

Topics: Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Curcumin; Cyclin D1; Cytochromes c; Female; Flow Cytometry; HSP70 Heat-Shock Proteins; Humans; Proto-Oncogene Proteins c-bcl-2; Telomerase; Uterine Cervical Neoplasms

Cervical cancer is the second most common malignant neoplasm in women, in terms of incidence and mortality rates worldwide, and is associated with excessive inflammation. This involves the expression of both pro- and anti-apoptotic proteins that have varied effect on tumor growth and metastasis. The objective of the present study was to elucidate the effect of hydrogen peroxide (H2O2) on apoptotic signal molecules in vitro in SiHa and CaSki cell lines expressing the human papilloma virus 16 E6 protein, which causes the ubiquitin-mediated degradation of p53 protein and is thus p53 deficient. The p53 is known to act as a cellular stress sensor and triggers apoptosis. We demonstrate, here, that in HPV 16 positive cell lines apoptosis is triggered by upregulation of p73, which causes activation of pro-apoptotic Bax accompanied by down regulation of anti-apoptotic Bcl xl, release of cytochrome c from mitochondria and activation of caspases-9 and -3.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; DNA-Binding Proteins; Down-Regulation; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; HSP70 Heat-Shock Proteins; Human papillomavirus 16; Humans; Hydrogen Peroxide; Mitochondria; Mitogen-Activated Protein Kinases; Nuclear Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins p21(ras); Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Up-Regulation; Uterine Cervical Neoplasms

We describe a new time alignment method that takes advantage of both dimensions of LC-MS data to resolve ambiguities in peak matching while remaining computationally efficient. This approach, Warp2D, combines peak extraction with a two-dimensional correlation function to provide a reliable alignment scoring function that is insensitive to spurious peaks and background noise. One-dimensional alignment methods are often based on the total-ion-current elution profile of the spectrum and are unable to distinguish peaks of different masses. Our approach uses one-dimensional alignment in time, but with a scoring function derived from the overlap of peaks in two dimensions, thereby combining the specificity of two-dimensional methods with the computational performance of one-dimensional methods. The peaks are approximated as two-dimensional Gaussians of varying width. This approximation allows peak overlap (the measure of alignment quality) to be calculated analytically, without computationally intensive numerical integration in two dimensions. To demonstrate the general applicability of Warp2D, we chose a variety of complex samples that have substantial biological and analytical variability, including human serum and urine. We show that Warp2D works well with these diverse sample sets and with minimal tuning of parameters, based on the reduced standard deviation of peak elution times after warping. The combination of high computational speed, robustness with complex samples, and lack of need for detailed tuning makes this alignment method well suited to high-throughput LC-MS studies.

Topics: Adult; Aged; Aged, 80 and over; Animals; Blood Proteins; Cytochromes c; Data Interpretation, Statistical; Female; Gas Chromatography-Mass Spectrometry; Horses; Humans; Middle Aged; Pregnancy; Urinalysis; Uterine Cervical Neoplasms

The mechanism of antitumor action of a synthetic nitroflavone derivative, 2'-nitroflavone, was evaluated in vitro in HeLa human cervix adenocarcinoma cells. We showed that the nitroflavone derivative slowed down the cell cycle at the S phase and increase the population of cells at the G2/M phase after 24h of incubation. The treatment with 2'-nitroflavone also induced an apoptotic response, characterized by an increase of the sub-G1 fraction of cells, by cells with chromatin condensation and membrane blebbing, by a typical ladder of DNA fragmentation and by detection of apoptotic cells stained with Annexin V. The observed apoptosis was regulated by caspase-8 and -9, both contributing to the activation of the effector caspase-3. In addition, inhibitors of caspase-8 or -9 partially protected HeLa cells from 2'-nitroflavone-induced cell death. We also found that 2'-nitroflavone did not affect the total amount of Bax and Bcl-2 proteins, although a translocation of Bax from cytosol to mitochondria was evident after 6h of exposure. Furthermore, 2'-nitroflavone decreased the expression of the anti-apoptotic Bcl-XL protein, induced the release of cytochrome C to cytosol and increased the levels of Fas and Fas-L. Our results indicated that both death receptor and mitochondria-dependent pathways are involved in the apoptotic cell death triggered by 2'-nitroflavone and suggest that this derivative could be a potentially useful agent for the treatment of certain malignancies.

Topics: Apoptosis; Caspases; Cell Cycle; Cytochromes c; Fas Ligand Protein; Female; Flavones; HeLa Cells; Humans; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Uterine Cervical Neoplasms

ACTX-8 is a protein isolated from Agkistrodon acutus snake venom in our laboratory. It demonstrates cytotoxic activity on various carcinoma cell lines in vitro. However, the mechanism by which ACTX-8 inhibits cell proliferation remains poorly understood. In this study the influence of ACTX-8 on the activation of apoptotic pathway in Hela cells was investigated. We demonstrated that cell death induced by ACTX-8 was concentration- and time-dependent. Apoptotic changes such as phosphatidyl serine externalization and DNA fragmentation were detected in ACTX-8-treated cells. Caspase activation and reactive oxygen species (ROS) production were involved in ACTX-8-induced apoptosis, but pan caspase inhibitor, z-VAD-fmk, could not inhibit cell death induced by ACTX-8 completely, which proved the existence of another pathway for ACTX-8-induced cell death. We found cytochrome c release into cytosol and mitochondrial membrane potential (MMP) dissipation in ACTX-8-treated cells, which indicated that mitochondrial pathway played a role in ACTX-8-induced cell apoptosis. The ratio of expression levels of pro- and anti-apoptotic Bcl-2 family members was not changed by ACTX-8 treatment. However Bad and Bax were translocated from cytosol into mitochondria, and the coimmunoprecipitation result indicated that in mitochondria Bak and Bcl-xL dissociation was followed by the binding of Bad and Bcl-xL. Taken together, the study indicated mitochondrial pathway played an important role in the ACTX-8-induced apoptosis, which was regulated by Bcl-2 family members.

Topics: Agkistrodon; Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Fractionation; Cell Survival; Crotalid Venoms; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Enzyme Activation; Enzyme Inhibitors; Female; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Phosphatidylserines; Proto-Oncogene Proteins c-bcl-2; Uterine Cervical Neoplasms

Liquid Chromatography--Mass Spectrometry (LC-MS) is a powerful method for sensitive detection and quantification of proteins and peptides in complex biological fluids like serum. LC-MS produces complex data sets, consisting of some hundreds of millions of data points per sample at a resolution of 0.1 amu in the m/z domain and 7000 data points in the time domain. However, the detection of the lower abundance proteins from this data is hampered by the presence of artefacts, such as high frequency noise and spikes. Moreover, not all of the tens of thousands of the chromatograms produced per sample are relevant for the pursuit of the biomarkers. Thus in analysing the LC-MS data, two critical pre-processing issues arise. Which of the thousands of the: 1. chromatograms per sample are relevant for the detection of the biomarkers?, and 2. signals per chromatogram are truly compound-related? Each of these issues involves assessing the significance (deviation from noise) of multiple observations and the issue of multiple comparisons arises. Current methods disregard the multiplicity and provide no concrete threshold for significance. However, with such procedures, the probability of one or more false-positives is high as the number of tests to be performed is large, and must be controlled. Realizing that the cut-offs for declaring a chromatogram (or a signal) to be compound-related can hugely influence which proteins are detected, it seems natural to define thresholds that are neither arbitrary nor subjective. We suggest the choice of thresholds guided by the critical aim of controlling the False Discovery Rate (FDR) in multiple hypotheses testing for significance over a large set of features produced per sample. This involves the use of the regression diagnostics to characterize the signals of a chromatogram (e.g. as outliers or influential) and to suggest suitable tests statistics for the multiple testing procedures (MTP) for discriminating noise and spikes from true signals. The role of the Generalized Linear Models (GLM) in this MTP is investigated. The method is applied to LC-MS datasets from trypsin-digested serum spiked with varying levels of horse heart cytochrome C (cytoc).

Topics: Algorithms; Animals; Artifacts; Biomarkers; Chromatography, Liquid; Cytochromes c; Female; Horses; Humans; Mass Spectrometry; Models, Theoretical; Myocardium; Proteome; Regression Analysis; Solvents; Uterine Cervical Neoplasms

To investigate the apoptosis-inducing effect of oridonin, a diterpenoid isolated from Rabdosia rubescens, in the human cervical carcinoma HeLa cell line.. A morphological analysis, nuclear condensation, and fragmentation of chromatin were monitored using Hoechst 33342 staining. Cell viability was assessed using the 3-(4, 5-dimethylthiazol-(2)-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cell apoptosis and the apoptosis-related activation in the HeLa cell line were evaluated by flow cytometry and Western blotting.. Oridonin suppressed the proliferation of the HeLa cell line in a dose- and time-dependent fashion. Oridonin treatment downregulated the activation of protein kinase B (Akt), the expression of forkhead box class O (FOXO) transcription factor, and glycogen synthase kinase 3 (GSK3). Oridonin also induced the release of cytochrome c accompanied by the activation of caspase-3 and poly-adenosine diphosphate- ribose polymerase cleavage. In addition, Z-D(OMe)-E(OMe)-V-D(OMe)- FMK (z-DEVD-fmk), an inhibitor of caspases, prevented caspase-3 activation and abrogated oridonin-induced cell death. Finally, oridonin treatment of the HeLa cell line downregulated the expression of the inhibitor of the apoptosis protein.. Our results showed that oridonin-induced apoptosis involved several molecular pathways. Oridonin may suppress constitutively activated targets of phosphatidylinositol 3-kinase (Akt, FOXO, and GSK3) in the HeLa cell line, inhibiting the proliferation and induction of caspase-dependent apoptosis.

Topics: Apoptosis; Caspases; Collagen Type XI; Cytochromes c; Diterpenes, Kaurane; Down-Regulation; Female; HeLa Cells; Humans; In Vitro Techniques; Membrane Potential, Mitochondrial; Mitochondria; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Uterine Cervical Neoplasms

Oncogenic types of human papillomaviruses (HPVs) cause cervical cancer in humans. The antiapoptotic viral E6 gene has been identified as a key factor for maintaining the viability of HPV-positive cancer cells. Although E6 has the potential to modulate many apoptosis regulators, the crucial apoptotic pathway blocked by endogenous E6 in cervical cancer cells remained unknown. Using RNA interference (RNAi), here, we show that targeted inhibition of E6 expression in cervical cancer cells leads to the transcriptional stimulation of the PUMA promoter, in a p53-dependent manner. This is linked to the activation and translocation of Bax to the mitochondrial membrane, cytochrome c release into the cytosol, and activation of caspase-3, in a PUMA-dependent manner. Moreover, inhibition of Bax expression by RNAi efficiently reverts the apoptotic phenotype, which results from inhibition of E6 expression. Thus, interference with the p53/PUMA/Bax cascade is crucial for the antiapoptotic function of the viral E6 oncogene in HPV-positive cancer cells.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Caspase 3; Caspases; Cytochromes c; DNA-Binding Proteins; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Mitochondria; Oncogene Proteins, Viral; Protein Transport; Proto-Oncogene Proteins; Repressor Proteins; RNA Interference; RNA, Small Interfering; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Proteomic studies have stimulated the development of novel stationary phases in miniaturised chromatographic columns that permit high linear flow velocities and exhibit high resolving power. In this work, a 50-microm reversed-phase silica-based monolith was chromatographically characterised for its use in proteomics applications using a nanoLC-MS set-up. It showed high efficiency for the separation of tryptic peptides under isocratic elution conditions (HETP(min)=5-10 microm at 2.4 mm/s). Flow rates up to 1.95 microL/min (18.4 mm/s) and gradient slopes up to an unusually fast 9% could be used. This resulted in rapid separations of peptide mixtures, with peak widths at half height of between 5 and 10 s. The 50-microm monolithic column was used to analyse depleted serum from a cervical cancer patient at a throughput of one sample per 30 min.

Topics: Amino Acid Sequence; Blood Proteins; Chromatography, High Pressure Liquid; Cytochromes c; Female; Humans; Molecular Sequence Data; Nanotechnology; Peptide Fragments; Proteomics; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Time Factors; Trypsin; Uterine Cervical Neoplasms

Early stage cervical cancer is treated with surgery or radiation with equivalent results. Radiation is used for curative therapy of locally advanced disease and is combined with additional anti-tumor agents to improve control. We determined the potential role of genistein as a radiosensitizer for cervical cancer cells.. Human cervical cell lines (CaSki and ME180) were used. Sensitivity of cells to genistein, radiation and the combination was determined by colony assays. Western blotting was used to study the expression of cell-response-related gene products.. Genistein results in the dose-dependent inhibition of all cell lines (2.5-40.0 microM). Effect of genistein on the radiosensitivity of the two cervical tumor cells was variable. Me180 cells were more sensitive at 20 and 40 microM of genistein. At 40 microM, less than 5% of Me180 cells survived the radiation (200-800 cGy). Potentiation of the radiation effect in CaSki cells was seen (500-800 cGy). The most significant enhancement of radiosensitivity was seen at 20 and 40 microM genistein at 500 and 800 cGy. G(2)M arrest was demonstrated only in ME180 cells with genistein. There was significant inhibition of Mcl-1 by genistein that correlated with increase in radiosensitivity in Me180 cells. Activated pAKT (Thr 308) was inhibited with genistein and radiation in CaSki cells. CONCLUSIONS.: Genistein inhibits growth of cervical cancer cells. Genistein results in variable and significant enhancement of the radiation effect that may be partially mediated by G(2)M arrest, Mcl-1 and activation of the AKT gene.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Combined Modality Therapy; Cytochromes c; Dose-Response Relationship, Drug; Female; Genistein; Humans; Isoflavones; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Radiation-Sensitizing Agents; Uterine Cervical Neoplasms

We have studied the effect of tri-phenyl tin benzimadazolethiolcopper chloride (TPT-CuCl(2)), a novel bimetallic compound, on the regulation of apoptosis in HeLa cells, MCF-7 cells, and in vivo Wistar rat model. TPT-CuCl(2) induces significant apoptosis in HeLa cell line characterized by DNA fragmentation and chromosome condensation. Comet assay revealed that TPT-CuCl(2) targets and causes severe damage to the DNA. Treatment of HeLa cells with TPT-CuCl(2) rescues the accumulation of p53 from the suppression of human papilloma virus E6, resulting in a dramatic up-regulation of Bax and Bak and down-regulation of the antiapoptotic factor Survivin. Apoptotic induction by TPT-CuCl(2) was shown to mediate in a p53-depedent manner; loss of p53 impairs the release of cytochrome c and Smac/DIABLO from mitochondria to cytosol. Moreover, we have shown that TPT-CuCl(2) induced-apoptosis was through an intrinsic mitochondrial pathway, which was inhibited by viral oncoprotein E1B19K. Caspase-3 was found to be indispensable in TPT-CuCl(2)-triggered apoptosis signaling pathway, because caspase-3 deficient cell line MCF-7 was resistant to TPT-CuCl(2). Furthermore, in vivo studies using C6 glioblastoma xenograft rat model revealed that TPT-CuCl(2) exhibits significant antiproliferative activity against tumor development with minimal cytotoxicity toward normal physiological function of the experimental rats. These findings imply the attractiveness of TPT-CuCl(2) as a drug candidate for further development.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Carrier Proteins; Caspase 3; Caspases; Cell Division; Cytochromes c; Down-Regulation; Enzyme Activation; Female; HeLa Cells; Humans; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Neoplasm Proteins; Organotin Compounds; Papillomaviridae; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Survivin; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

There is an emerging evidence that plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) may have potential as a chemotherapeutic agent. However, the growth inhibitory mechanisms of plumbagin have remained unexplored. The aim of the study was to determine whether plumbagin-induced cell death in human cervical cancer cell line, ME-180, exhibited biochemical characteristics of apoptosis and to check whether N-acetyl-l-cysteine (NAC), which is a free radical scavenger, can reverse the cytotoxic effects of plumbagin. It can be concluded from the results that plumbagin inhibits the growth of ME-180 cells in a concentration and time-dependent manner. The cytotoxic effect of plumbagin induced cell death is through the generation of reactive oxygen species (ROS) and subsequent induction of apoptosis as demonstrated by the present data. Treatment of cells with plumbagin caused loss of mitochondrial membrane potential (DeltaPsi(m)), and morphological changes characteristic of apoptosis, such as the translocation of phosphatidyl serine, nuclear condensation, and DNA fragmentation. Moreover, plumbagin-induced apoptosis involved release of mitochondrial cytochrome c and apoptosis inducing factor (AIF), thus activation of caspase-dependent and -independent pathways, as shown by the plumbagin-mediated activation of caspase-3 and -9. Our results also show that pretreatment of ME-180 cells with NAC blocks plumbagin-induced loss of DeltaPsi(m) and subsequent release of cytochrome c, AIF, and caspase-9 and -3 activation, thus inhibiting the apoptotic ability of plumbagin.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspases; Cell Nucleus; Cytochromes c; Dose-Response Relationship, Drug; Female; Humans; Mitochondria; Naphthoquinones; Phosphatidylserines; Reactive Oxygen Species; Time Factors; Uterine Cervical Neoplasms

Hyperthermia-induced apoptosis and its enhancement in the presence of a temperature-dependent free radical initiator, 2,2'-azobis (2-aminopropane) dihydrochloride (AAPH) were examined in human uterine cervical cancer cell lines, CaSki and HeLa. When both cell lines were treated with hyperthermia at 44 degrees C for 60 min, minimal apoptosis was observed. When combined with nontoxic AAPH (50mM), significant enhancement of apoptosis was observed, where the initial rate of free radical formation was about twice as high than that at 37 degrees C. Augmentation of the growth delay, lipid peroxidation (LPO), activation of caspase-3 and increase in [Ca2+]i were also observed after the combined treatment. A water-soluble vitamin E, Trolox, blocked the increase in [Ca2+]i and an intracellular Ca2+ chelator, BAPTA-AM, prevented the DNA fragmentation induced by the combination. Cytochrome c release was also revealed by fluorescence microscopy. However, no significant change in mitochondrial membrane potential and expression of Bax and Bcl-2 was observed. A slight increase in Fas expression was observed only in CaSki cells after the combined treatment. These results indicate that hyperthermia and AAPH induce enhanced apoptosis and subsequent cell killing via two pathways; a pathway dependenton increase in LPO and [Ca2+]i, and a pathway associated with cytochrome c release and subsequent caspase activation without changes of mitochondrial membrane potential and Bax/Bcl-2 expression in these cell lines. Since it is known that cancer cells are generally resistant to physical and chemical stress-induced apoptosis, free radical generators like AAPH appear to be a useful thermosensitizer for hyperthermic cancer therapy.

Topics: Amidines; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cytochromes c; DNA Fragmentation; Egtazic Acid; fas Receptor; Female; Fever; Flow Cytometry; Free Radicals; HeLa Cells; Humans; Ions; Lipid Peroxidation; Microscopy, Fluorescence; Oxidants; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Temperature; Time Factors; Uterine Cervical Neoplasms

The aim of the therapy of human malignancies is the inhibition of cell proliferation and/or induction of apoptosis. We studied the kinetics of the morphological and biochemical changes in HeLa cells during chemotherapy by cisplatin (CP). Apoptosis was evaluated by scoring of cells exhibiting changes characteristic for early and late stages of apoptosis as determined by Hoechst 33258 staining and by examination of positive reaction for activated caspase-3. Expression and intracellular localization of distinct proteins was analyzed by immunoblotting of subcellular fractions and segregation of nucleoli by immunocytochemistry. Chromatin fragmentation characteristic for apoptosis was observed in single cells after 3h cisplatin. A strong cytoplasmic accumulation of cytochrome C detected by immunoblotting 6h post-treatment was accompanied by an activation of caspase-9. Neither inhibition of cell division nor blocking of DNA replication preceded the onset of apoptosis. Our results show that after short treatment by CP, cell proliferation and apoptosis concomitantly occurred.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Nucleolus; Cisplatin; Cytochromes c; Female; Flow Cytometry; HeLa Cells; Humans; Immunohistochemistry; Uterine Cervical Neoplasms