cytochrome-c-t and Esophageal-Neoplasms

Barrett's esophagus (BE) is associated with reflux and is implicated the development of esophageal adenocarcinoma (EAC). Apoptosis induces cell death through mitochondrial outer membrane permeabilization (MOMP), which is considered an irreversible step in apoptosis. Activation of MOMP to levels that fail to reach the apoptotic threshold may paradoxically promote cancer-a phenomenon called "Minority MOMP." We asked whether reflux-induced esophageal carcinogenesis occurred via minority MOMP and whether compensatory resistance mechanisms prevented cell death during this process. We exposed preneoplastic, hTERT-immortalized Barrett's cell, CP-C and CP-A, to the oncogenic bile acid, deoxycholic acid (DCA), for 1 year. Induction of minority MOMP was tested via comet assay, CyQuant, annexin V, JC-1, cytochrome C subcellular localization, caspase 3 activation, and immunoblots. We used bcl-2 homology domain-3 (BH3) profiling to test the mitochondrial apoptotic threshold. One-year exposure of Barrett's cells to DCA induced a malignant phenotype noted by clone and tumor formation. DCA promoted minority MOMP noted by minimal release of cytochrome C and limited caspase 3 activation, which resulted in DNA damage without apoptosis. Upregulation of the antiapoptotic protein, Mcl-1, ROS generation, and NF-κB activation occurred in conjunction with minority MOMP. Inhibition of ROS blocked minority MOMP and Mcl-1 upregulation. Knockdown of Mcl-1 shifted minority MOMP to complete MOMP as noted by dynamic BH3 profiling and increased apoptosis. Minority MOMP contributes to DCA induced carcinogenesis in preneoplastic BE. Mcl-1 provided a balance within the mitochondria that induced resistance complete MOMP and cell death. Targeting Mcl-1 may be a therapeutic strategy in EAC.

Topics: Apoptosis; Barrett Esophagus; Bile Acids and Salts; Carcinogenesis; Cell Line; Cell Membrane Permeability; Cytochromes c; DNA Damage; Esophageal Neoplasms; Esophagus; Gastrointestinal Agents; Humans; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Signal Transduction

α-Hederin has been shown promising anti-tumor potential against various cancer cell lines. However, reports about effects of α-hederin on esophageal squamous cell carcinoma (ESCC) are still unavailable.. To investigate the inhibitory effects of α-hederin on ESCC and explore the underlying mechanism.. Human esophageal carcinoma cell line (Eca-109) was used for the experiment. Cell Counting Kit-8, flow cytometry, Hoechst 33258 staining, enhanced ATP assay kit, 2',7'-dichlorofluorescin diacetate, JC-1 kit, and Western bolt were used to assess the cell viability, cycle, apoptosis, cellular ATP content, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. Xenografted tumor model was constructed to evaluate the in vivo anti-tumor effects of α-hederin.. Compared with control group, α-hederin significantly inhibited the proliferation, induced apoptosis of ESCC, and arrested the cell cycle in G1 phase (P < 0.05). α-Hederin induced the accumulation of ROS, decrement of ATP levels, and disruption of MMP (P < 0.05). The detection of mitochondrial and cytosol proteins showed that AIF, Apaf-1, and Cyt C were released and increased in cytoplasm, and then, caspase-3, caspase-9, and Bax were involved and increased, while Bcl-2 level was decreased (P < 0.05). Furthermore, the above changes were amplified in the group pretreated with L-buthionine sulfoximine, while N-acetyl-L-cysteine plays an opposite role (P < 0.05). Meanwhile, α-hederin significantly inhibited the growth of xenografted tumors with favorable safety.. α-Hederin could inhibit the proliferation and induce apoptosis of ESCC via dissipation of the MMP with simultaneous ROS generation and activation of the mitochondrial pathway.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Apoptosis Inducing Factor; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Flow Cytometry; Humans; In Situ Nick-End Labeling; In Vitro Techniques; Male; Membrane Potential, Mitochondrial; Mice, Nude; Mitochondria; Neoplasm Transplantation; Oleanolic Acid; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Saponins; Xenograft Model Antitumor Assays

First generation silver(I) phosphines have garnered much interest due to their vast structural diversity and promising anticancer activity. Increasing incidences of cancer, side-effects to chemotherapeutic agents and redevelopment of tumors due to resistance prompts the exploration of alternative compounds showing anticancer activity. This study revealed the effective induction of cell death by a silver(I) thiocyanate 4-methoxyphenyl phosphine complex in a malignant esophageal cell line. Apoptotic cell death was confirmed in treated cells. Moreover, mitochondrial targeting via the intrinsic cell death pathway was evident due to low levels of ATP, altered ROS activity, mitochondrial membrane depolarization, cytochrome c release and caspase-9 cleavage. The complex displayed low cytotoxicity towards two human non-malignant, skin and kidney, cell lines. The findings reported herein give further insight into the selective targeting of silver(I) phosphines and support our belief that this complex shows great promise as an effective chemotherapeutic drug.

Topics: Adenosine Triphosphate; Apoptosis; Caspase 9; Cell Line, Tumor; Coordination Complexes; Cytochromes c; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Phosphines; Reactive Oxygen Species; Silver; Thiocyanates

Previous studies have shown that Tanshinone (Tan) IIA exerts obvious antitumor efficacy; however, its molecular mechanism remains unclear. This study was conducted to identify the influence of Tan IIA on Eca-109 cell apoptosis, explore its molecular mechanism, and provide a theoretical basis for clinical application.. Eca-109 cells were cultured in vitro and treated with different concentrations of Tan IIA. Morphologic changes were viewed under inverted fluorescence microscope with dual acridine orange/ethidium bromide staining assay. Methyl-thiazolyl-tetrazolium and Annexin V propidium iodide assays were respectively used to measure cell viability and apoptosis rate. The protein and messenger (m)RNA expression of binding immunoglobulin protein (BIP), mitochondrial cytochrome c (CytC), and caspase-9 were detected by Western blot and quantitative real-time PCR, respectively.. Cell viability decreased and the apoptosis rate significantly increased with increasing concentrations of Tan IIA (0, 20, 40, 60 μg/mL), which indicated that Tan IIA inhibited the proliferation and induced the apoptosis of Eca-109 cells in a dose-dependent manner. Eca-109 cells treated with 60 μg/mLTan IIA showed typical morphological changes of apoptosis under the inverted microscope. Moreover, compared with the negative control group, protein and mRNA expression of BIP decreased significantly (P < 0.05), whereas protein and mRNA expression of CytC and caspase-9 increased significantly (P < 0.05).. Tan IIA can induce apoptosis in human esophageal carcinoma Eca-109 cells by regulating BIP, CytC, and caspase-9 expression. Endoplasmic reticulum stress and mitochondrial-dependent may be involved in Tan IIA-induced Eca-109 cell apoptosis.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Signal Transduction

Gold nanoparticles (AuNP's) facilitate cancer cell recognition and can be manufactured by green synthesis using nutrient rich medicinal plants such as Moringa oleifera (MO). Targeting dysregulated oncogenes and tumor suppressor genes is crucial for cancer therapeutics. We investigated the antiproliferative effects of AuNP synthesized from MO aqueous leaf extracts (MLAuNP ) in A549 lung and SNO oesophageal cancer cells. A one-pot green synthesis technique was used to synthesise MLAuNP . A549, SNO cancer cells and normal peripheral blood mononuclear cells (PBMCs) were exposed to MLAuNP and CAuNP to evaluate cytotoxicity (MTT assay); apoptosis was measured by phosphatidylserine (PS) externalization, mitochondrial depolarization (ΔΨm) (flow cytometry), caspase-3/7, -9 activity, and ATP levels (luminometry). The mRNA expression of c-myc, p53, Skp2, Fbw7α, and caspase-9 splice variants was determined using qPCR, while relative protein expression of c-myc, p53, SRp30a, Bax, Bcl-2, Smac/DIABLO, Hsp70, and PARP-1 were determined by Western blotting. MLAuNP and CAuNP were not cytotoxic to PBMCs, whilst its pro-apoptotic properties were confirmed in A549 and SNO cells. MLAuNP significantly increased caspase activity in SNO cells while MLAuNP significantly increased PS externalization, ΔΨm, caspase-9, caspase-3/7 activities, and decreased ATP levels in A549 cells. Also, p53 mRNA and protein levels, SRp30a (P = 0.428), Bax, Smac/DIABLO and PARP-1 24 kDa fragment levels were significantly increased. Conversely, MLAuNP significantly decreased Bcl-2, Hsp70, Skp2, Fbw7α, c-myc mRNA, and protein levels and activated alternate splicing with caspase-9a splice variant being significantly increased. MLAuNP possesses antiproliferative properties and induced apoptosis in A549 cells by activating alternate splicing of caspase-9. J. Cell. Biochem. 117: 2302-2314, 2016. © 2016 Wiley Periodicals, Inc.

Topics: A549 Cells; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspase 9; Cell Proliferation; Cytochromes c; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Gold; Humans; Leukocytes, Mononuclear; Lung Neoplasms; Male; Metal Nanoparticles; Moringa oleifera; Oncogenes; Plant Extracts; RNA Splicing; Tumor Cells, Cultured

A ruthenium(II) complex [Ru(p-cymene)(NHC)Cl2] (NHC=1,3-bis(4-(tert-butyl)benzylimidazol-2-ylidene), referred to as L-4, has been designed and synthesized recently in order to look for new anticancer drugs with high efficacy and low side effects. The anticancer activity and mechanism of action of L-4 in human esophageal squamous carcinoma EC109 cells were systematically investigated. The results revealed that L-4 exerted strong inhibitory effect on the proliferation of EC109 cells, and it arrested EC109 cells at G2/M phase, accompanied with the up-regulation of p53 and p21 and the down-regulation of cyclin D1. The results also showed that the reactive oxygen species (ROS)-dependent apoptosis of EC109 can be induced by L-4 via inhibiting the activity of glutathione reductase (GR), decreasing the ratio of glutathione to oxidized glutathione (GSH/GSSG), and leading to the generation of reactive oxygen species. The mitochondria-mediated apoptosis of EC109 induced by L-4 was also observed from the increase of Bax/Bcl-2 ratio, overload of Ca(2+), disruption of mitochondrial membrane potential (MMP), redistribution of cytochrome c, and activation of caspase-3/-9. However, the effects of L-4 on the cell viability, GR activity, GSH/GSSG ratio, reactive oxygen species level, mitochondria dysfunction and apoptosis induction were remarkably attenuated by adding the reactive oxygen species scavenger, NAC. Therefore, it was concluded that L-4 can inhibit the proliferation of EC109 cells via blocking cell cycle progression and inducing reactive oxygen species-dependent and mitochondria-mediated apoptosis. These findings suggested that the ruthenium(II) complex might be a potential effective chemotherapeutic agent for human esophageal squamous carcinoma (ESCC) and worthy of further investigation.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Calcium; Carcinoma, Squamous Cell; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Humans; Intracellular Space; Membrane Potential, Mitochondrial; Models, Molecular; Molecular Conformation; Organometallic Compounds; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Ruthenium

Ent-kaurane diterpene compounds have attracted considerable attention in recent years due to its antitumor, antibacterial, and antiviral activities. However, the clinical development of natural kaurane diterpenes, for example, oridonin for cancer therapy has been hampered by its relatively moderate potency, limited bioavailability. Herein, we report a newly synthetic analog of natural ent-kaurane diterpene, DS2, which exhibits significantly improved activity of antiproliferation against various cancer cell lines relative to oridonin. DS2 treatment triggers the mitochondria-mediated apoptosis and cell cycle arrest in human esophageal cancer cell lines (EC9706, EC109). Interestingly, normal human esophageal epithelial cells (HEECs) and normal human liver cells (HL-7702) are both significantly more resistant to the growth inhibition by DS2 compared with esophageal cancer cells. The DS2-induced apoptosis in EC9706 cells correlated with the drop of mitochondrial membrane potential (MMP), release of cytochrome c into the cytosol and activation of caspase-9 and -3. The induction of proapoptotic proteins p21 and Bax were also observed in DS2-treated cells. The DS2-induced apoptosis was significantly attenuated by knockdown of Bax proteins. Meanwhile, the DS2 treatment caused generation of reactive oxygen species (ROS) in human esophageal cancer cells, but not in HEECs, which was attenuated by pretreatment with ROS scavenger N-acetylcysteine (NAC). More interestingly, the antioxidants pretreatment completely attenuated DS2 mediated loss of the MMP and apoptosis, as well as Bax expression and growth inhibition. In conclusion, the present study reveals that the mitochondria-mediated cell death by DS2 is associated with Bax regulation and ROS generation, and understanding the function and mechanism of DS2 will help us to design better anti-cancer drugs.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Diterpenes, Kaurane; Esophageal Neoplasms; Humans; Mitochondria; Reactive Oxygen Species

Matrine, as a member of Sophora family, is an alkaloid found in plants, and produces plethora pharmacological effects, including anti-cancer effects. However, the mechanism involved remains largely unknown. This study is conducted to investigate the anti-cancer mechanisms of matrine in human esophageal cancer in vitro and in vivo. In human esophageal cancer cell Eca-109, matrine significantly decreased the cell viability in a dose-dependent manner, and induced apoptosis as well as cell cycle arrest in G0/G1 phase by up-regulation of P53 and P21. The expression of several apoptosis-related proteins in cells and tumor tissues were evaluated by Western blot analysis. We found that matrine induced cell apoptosis by down-regulation of the ratio of BCL-2/BID and increasing activation of caspase-9. Further studies indicated that matrine induced apoptosis of Eca-109 was through the mitochondria-mediated internal pathway, but not by death receptor-mediated extrinsic apoptotic pathway, which was confirmed by the fact that Bid translocated from the nucleus to mitochondria during the process of the apoptosis induced by matrine. In vivo study found that matrine effectively inhibited the tumor formation of Eca-109 cells in nude mice. Our study suggests that matrine could serve as a potential novel agent from natural products to treat esophageal cancer.

Topics: Alkaloids; Animals; Antineoplastic Agents; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caspase 8; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Disease Models, Animal; Esophageal Neoplasms; Humans; Male; Matrines; Mice; Mitochondria; Models, Biological; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Quinolizines; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

OSU-03012 is a celecoxib derivative devoid of cyclooxygenase-2 inhibitory activity. It was previously reported to inhibit the growth of some tumor cells through the AKT-signaling pathway. In the current study, we assessed the ability of OSU-03012 to induce apoptosis in human esophageal carcinoma cells and the mechanism by which this occurs. A cell proliferation assay indicated that OSU-03012 inhibited the growth of human esophageal carcinoma cell lines with an IC50 below 2 μmol/l and had the most effective cytotoxicity against Eca-109 cells. Terminal deoxynucleotidyl transferase-mediated nick-end labeling assay and flow cytometry analysis showed that OSU-03012 could induce the apoptosis in Eca-109 cells. After treatment of Eca-109 cells with 2 μmol/l OSU-03012 for 24 h, the apoptosis index increased from 14.07 to 53.72%. OSU-03012 treatment resulted in a 30-40% decrease in the mitochondrial membrane potential and caused cytochrome c release into the cytosol. Further studies with caspase-9-specific and caspase-8-specific inhibitors (z-LEHDfmk and z-IETDfmk, respectively) pointed toward the involvement of the caspase-9 pathway, but not the caspase-8 pathway, in the execution of OSU-03012-induced apoptosis. Immunoblot analysis demonstrated that OSU-03012-induced cellular apoptosis was associated with upregulation of Bax, cleaved caspase-3, and cleaved caspase-9. Ser-15 of p53 was phosphorylated after 24 h of treatment of the cancer cells with OSU-03012. This increase in p53 was associated with the decrease in Bcl-2 and increase in Bax. An inhibitor of p53, pifithrin-α, attenuated the anticancer effects of OSU-03012 and downregulated the expression of Bax and cleaved caspase-9. Altogether, our results show that OSU-03012 could induce apoptosis in human esophageal carcinoma cells through a p53/Bax/cytochrome c/caspase-9-dependent pathway.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 9; Celecoxib; Cell Line, Tumor; Cytochromes c; Esophageal Neoplasms; Humans; Pyrazoles; Signal Transduction; Sulfonamides; Tumor Suppressor Protein p53

Drugs in clinical use with indole structure exhibit side effects. Therefore, to search for indole compounds with more efficacy and less side effect for cancer therapy, we developed a novel indole compound SK228 and examined its effects and mechanisms on antitumor growth and invasion inhibition in cell and tumor xenografts in nude mice models. SK228 significantly inhibited growth of different lung and esophageal cancer cell lines at sub-micromolar range, but not normal lung cells. SK228 induced DNA damages mainly by producing reactive oxygen species (ROS) resulting in apoptosis. SK228 treatment increased the release of cytochrome c into the cytosol along with the increased activity of caspase-3 and -9 without affecting caspase-8, whereas these effects were attenuated by ROS inhibitor. The expression levels of BCL-2 family regulators were also affected. Moreover, low-dose SK228 significantly reduced the invasion of cancer cells. The active phosphorylated form of FAK/Paxillin signaling pathway proteins and active form of RhoA were decreased. Moreover, the F-actin cytoskeleton was disrupted after low-dose SK228 treatment. Growth of an A549 tumor cell xenograft was markedly inhibited without significant side effects. SK228-induced apoptosis was confirmed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry of cleaved caspase-3 in tumors from treated mice. Our study provides the first evidence that SK228 exhibits cancer cell-specific cytotoxicity by inducing mitochondria-mediated apoptosis. In addition, SK228 inhibits cancer cell invasion via FAK/Paxillin disruption at noncytotoxic doses. SK228 can be further tested as a pharmaceutical compound for cancer treatment.

Topics: Actin Cytoskeleton; Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Damage; Esophageal Neoplasms; Focal Adhesion Protein-Tyrosine Kinases; Humans; Indoles; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Neoplasm Invasiveness; Neoplasms, Experimental; Paxillin; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; rhoA GTP-Binding Protein; Signal Transduction; Xenograft Model Antitumor Assays; Xylenes

Cisplatin (DDP)-based adjuvant chemotherapy is widely used for the treatment of esophageal cancer. However, DDP resistance has become more common and thus new approaches are required to be explored. Cisplatin was used to induce autophagy in the human esophageal cancer cell line, EC9706 cells, and the effect of autophagy on the survival of EC9706 cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by CCK8 assay. Apoptosis and cell cycle were detected by flow cytometry. Monodansylcadaverine (MDC) was used to detect autophagy. Western blotting assay was used to investigate the molecular changes that occurred in the course of treatment. DDP inhibited cell proliferation, induced cell death and cell cycle arrest at S phage. Moreover, autophagy was activated through class III PI3K pathway. The expression of autophagy-related Beclin1 and LC3-I was up-regulated and part of LC3-I was converted into LC3-II. However, after the combination treatment of 3-MA and DDP, the cell inhibitory rate increased; the apoptosis rate and the numbers of cells in S phase also increased. Furthermore, the accumulation of autophagic vacuoles was decreased; the expression of Beclin1 and LC3 was significantly down-regulated and the release of cytochrome c was decreased. DDP-induced apoptosis in EC9706 cells can be enhanced by the inhibitor of autophagy, 3-MA. Autophagy might play a role as a self-protective mechanism in DDP-treated esophageal cancer cells, and its inhibition could be a novel strategy for the adjuvant chemotherapy of esophageal cancer.

Topics: Adenine; Antineoplastic Agents; Apoptosis; Autophagy; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Proliferation; Cisplatin; Cytochromes c; Drug Synergism; Esophageal Neoplasms; Flow Cytometry; Humans; Tumor Cells, Cultured

Second mitochondria-derived activator of caspase (Smac) regulates chemotherapy-induced apoptosis. Smac mimetics have been tested in clinical trials as chemosensitizers. We determined the role of Smac in modulating the chemosensitivity of esophageal squamous cell carcinoma (ESCC).. Smac expression was evaluated in tissues from ESCC patients with differential chemotherapeutic responses. The effects of Smac knockdown and Smac mimetics on the chemosensitivity of ESCC cells and the molecular mechanisms by which Smac and Smac mimetics modulate chemosensitivity were determined. The therapeutic responses of ESCC cells with different Smac statuses were compared using xenograft models.. We found that Smac was significantly downregulated in most ESCC samples (36.8%, 25/68, P = 0.001), and Smac expression differed significantly (P < 0.05) between chemosensitive and chemoresistant tumors. The associations of tested factors and their responses were examined using logistic regression analysis. In ESCC cells treated with cisplatin, a common chemotherapeutic drug, Smac and cytochrome c were released from mitochondria, and caspase-3 and caspase-9 were activated. Knockdown of Smac abrogated cisplatin-induced apoptosis, mitochondrial dysfunction, cytochrome c release, and caspase activation. Smac deficiency also reduced the effect of cisplatin on long-term cell viability, and led to cisplatin resistance in xenograft tumors in vivo. LBW242, a small molecule Smac mimetic, enhanced cisplatin-induced apoptosis and caspase activation and restored cisplatin sensitivity in Smac-deficient cells.. Our data suggested that downregulation of Smac may be a chemoresistance mechanism in ESCC. Combinations of Smac mimetics with chemotherapeutic agents may have therapeutic benefits for the treatment of esophageal cancer.

Topics: Adult; Aged; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Squamous Cell; Caspases; Cell Line, Tumor; Cisplatin; Cytochromes c; Down-Regulation; Drug Resistance, Neoplasm; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Logistic Models; Male; Mice; Mice, Nude; Middle Aged; Mitochondrial Proteins; Oligopeptides; RNA Interference; Treatment Outcome; Xenograft Model Antitumor Assays

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

Upper gastrointestinal adenocarcinomas are a common cause of cancer-related deaths. In this study, the authors investigated the prevalence and biological significance of Aurora Kinase A (AURKA) overexpression in upper gastrointestinal adenocarcinomas.. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining on tumor tissue microarrays (TMA) were used to study the expression of AURKA in upper gastrointestinal adenocarcinomas. To investigate the biological and signaling impact of AURKA, the authors used multiple in vitro assays that included 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling), cytochrome C release, flow cytometry, luciferase reporter, and Western blot analysis.. Frequent overexpression of AURKA transcript in upper gastrointestinal adenocarcinomas was detected compared with normal samples (47%; P= .001). The immunohistochemical analysis of 130 tumors demonstrated moderate-to-strong immunostaining of AURKA in >50% of upper gastrointestinal adenocarcinomas. By using camptothecin as a drug-induced apoptosis in vitro model, the authors demonstrated that the expression of AURKA provided protection against apoptosis to gastrointestinal cancer cells (AGS and RKO) (P= .006) and RIE-1 primary intestinal epithelial cells (P= .001). The AURKA overexpression mediated an increase in phosphorylation of AKT(Ser473) with an increase in HDM2 level. The shRNA-knockdown of AKT in AURKA-overexpressing cells reversed this effect and showed a significant increase in the p53 protein level, indicating a possible nexus of AURKA/AKT/p53. Indeed, overexpression of AURKA led to a remarkable reduction in the transcription activity of p53, with subsequent reductions in transcript and protein levels of its downstream proapoptotic transcription targets (p21, BAX, NOXA, and PUMA).. Study results indicated that AURKA provides potent antiapoptotic properties to gastrointestinal cells by regulating levels of p53 through the AKT/HDM2 axis.

Topics: Adenocarcinoma; Apoptosis; Aurora Kinase A; Aurora Kinases; Biomarkers, Tumor; Blotting, Western; Camptothecin; Coloring Agents; Cytochromes c; Enzyme Inhibitors; Esophageal Neoplasms; Flow Cytometry; Gene Expression Regulation, Enzymologic; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Luciferases; Luminescent Agents; Polymerase Chain Reaction; Protein Array Analysis; Protein Serine-Threonine Kinases; Stomach Neoplasms; Tetrazolium Salts; Thiazoles; Tumor Suppressor Protein p53

Gastroduodenal reflux is implicated in esophageal carcinogenesis. This effect is mediated by reactive oxygen species. We hypothesized that this is mediated by DNA mismatch lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG), which is repaired by the Mut Y homologue (MYH). We tested the effect of reflux, either alone or in combination with the human dietary mutagen methyl-n-amyl nitrosamine (MNAN), on DNA damage in adenocarcinoma and squamous cell cancer of the esophagus in a rat model.. Reflux was promoted in male Sprague-Dawley rats by duodenoesophageal anastomosis (8 weeks) without gastric bypass. MNAN treatment (25 mg/kg per week intraperitoneally for four doses) commenced at 10 weeks age. Ten animals served as controls. Quantification of 8-oxoG was performed by using immunohistochemistry, and MYH was analyzed by Western blot. Apoptosis was assessed by terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL), cytochrome C, and caspase.. Tumors (adenocarcinoma) developed in 15 (50%) of 30 animals with reflux alone; this increased to 26 (86.6%) of 30 when reflux was combined with MNAN treatment, with tumor histology consistent with adenosquamous and squamous cell cancer. DNA damage, as reflected by positive 8-oxoG staining in reflux groups, was significantly increased compared with control (p < 0.01), and this was maximal in tissues with malignant transformation. Protein levels of the DNA repair enzyme MYH were significantly less in tissues subjected to reflux compared with controls (p < 0.05). TUNEL, cytochrome C, and caspase positivity confirmed increased apoptosis in cancer lesions.. Gastroduodenal reflux leads to increased DNA damage and downregulation of the DNA mismatch repair pathway. This pathway has an important role in esophageal carcinogenesis in rats.

Topics: Adenocarcinoma; Animals; Apoptosis; Carcinogens; Carcinoma, Squamous Cell; Caspases; Cytochromes c; DNA Damage; DNA Mismatch Repair; DNA Repair Enzymes; Down-Regulation; Duodenogastric Reflux; Esophageal Neoplasms; Guanosine; In Situ Nick-End Labeling; Male; Nitrosamines; Rats; Rats, Sprague-Dawley; Staining and Labeling