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Dichloroacetate affects proliferation but not apoptosis in canine mammary cell lines.

ABSTRACT: Targeting mitochondrial energy metabolism is a novel approach in cancer research and can be traced back to the description of the Warburg effect. Dichloroacetate, a controversially discussed subject of many studies in cancer research, is a pyruvate dehydrogenase kinase inhibitor. Dichloroacetate causes metabolic changes in cancerous glycolysis towards oxidative phosphorylation via indirect activation of pyruvate dehydrogenase in mitochondria. Canine mammary cancer is frequently diagnosed but after therapy prognosis still remains poor. In this study, canine mammary carcinoma, adenoma and non-neoplastic mammary gland cell lines were treated using 10 mM Dichloroacetate. The effect on cell number, lactate release and PDH expression and cell respiration was investigated. Further, the effect on apoptosis and several apoptotic proteins, proliferation, and microRNA expression was evaluated. Dichloroacetate was found to reduce cell proliferation without inducing apoptosis in all examined cell lines.

SUBMITTER: Harting TP

PROVIDER: S-EPMC5462399 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Dichloroacetate affects proliferation but not apoptosis in canine mammary cell lines.

Harting Tatjana P TP Stubbendorff Mandy M Hammer Susanne C SC Schadzek Patrik P Ngezahayo Anaclet A Murua Escobar Hugo H Nolte Ingo I

PloS one 20170607 6

Targeting mitochondrial energy metabolism is a novel approach in cancer research and can be traced back to the description of the Warburg effect. Dichloroacetate, a controversially discussed subject of many studies in cancer research, is a pyruvate dehydrogenase kinase inhibitor. Dichloroacetate causes metabolic changes in cancerous glycolysis towards oxidative phosphorylation via indirect activation of pyruvate dehydrogenase in mitochondria. Canine mammary cancer is frequently diagnosed but aft ...[more]

PMID: 28591165

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Project description:BackgroundDoxorubicin (DOX) is widely used in both human and veterinary oncology although the onset of multidrug resistance (MDR) in neoplastic cells often leads to chemotherapy failure. Better understanding of the cellular mechanisms that circumvent chemotherapy efficacy is paramount. The aim of this study was to investigate the response of two canine mammary tumour cell lines, CIPp from a primary tumour and CIPm, from its lymph node metastasis, to exposure to EC50(20h) DOX at 12, 24 and 48 h of treatment. We assessed the uptake and subcellular distribution of DOX, the expression and function of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), two important MDR mediators. To better understand this phenomenon the effects of DOX on the cell cycle and Ki67 cell proliferation index and the expression of p53 and telomerase reverse transcriptase (TERT) were also evaluated by immunocytochemistry (ICC).ResultsBoth cell lines were able to uptake DOX within the nucleus at 3 h treatment while at 48 h DOX was absent from the intracellular compartment (assessed by fluorescence microscope) in all the surviving cells. CIPm, originated from the metastatic tumour, were more efficient in extruding P-gp substrates. By ICC and qRT-PCR an overall increase in both P-gp and BCRP were observed at 48 h of EC50(20h) DOX treatment in both cell lines and were associated with a striking increase in the percentage of p53 and TERT expressing cells by ICC. The cell proliferation fraction was decreased at 48 h in both cell lines and cell cycle analysis showed a DOX-induced arrest in the S phase for CIPp, while CIPm had an increase in cellular death without arrest. Both cells lines were therefore composed by a fraction of cells sensible to DOX that underwent apoptosis/necrosis.ConclusionsDOX administration results in interlinked modifications in the cellular population including a substantial effect on the cell cycle, in particular arrest in the S phase for CIPp and the selection of a subpopulation of neoplastic cells bearing MDR phenotype characterized by P-gp and BCRP expression, TERT activation, p53 accumulation and decrease in the proliferating fraction. Important information is given for understanding the dynamic and mechanisms of the onset of drug resistance in a neoplastic cell population.

Project description:BACKGROUND: Ghrelin is a natural ligand of the growth hormone secretagogue receptor (GHS-R). They are often co-expressed in multiple human tumors and related cancer cell lines what can indicate that the ghrelin/GHS-R axis may have an important role in tumor growth and progression. However, a role of ghrelin in canine tumors remains unknown. Thus, the aim of our study was two-fold: (1) to assess expression of ghrelin and its receptor in canine mammary cancer and (2) to examine the effect of ghrelin on carcinoma cells proliferation, apoptosis, migration and invasion. The expression of ghrelin and its receptor in canine mammary cancer tissues and cell lines (isolated from primary tumors and their metastases) was examined using Real-time qPCR and immunohistochemistry. For apoptosis analysis the Annexin V and propidium iodide dual staining was applied whereas cell proliferation was evaluated by MTT assay and BrdU incorporation test. The influence of ghrelin on cancer cells migration and invasion was assessed using Boyden chamber assays and wound healing assay. RESULTS: The highest expression of ghrelin was observed in metastatic cancers whereas the lowest expression of ghrelin receptor was detected in tumors of the 3rd grade of malignancy. Higher expression of ghrelin and its receptor was detected in cancer cell lines isolated from metastases than in cell lines isolated from primary tumors. In vitro experiments demonstrated that exposure to low doses of ghrelin stimulates cellular proliferation, inhibits apoptosis and promotes motility and invasion of canine mammary cancer cells. Growth hormone secretagogue receptor inhibitor ([D-Lys3]-GHRP6) as well as RNA interference enhances early apoptosis. CONCLUSION: The presence of ghrelin and GHS-R in all of the examined canine mammary tumors may indicate their biological role in cancer growth and development. Our experiments conducted in vitro confirmed that ghrelin promotes cancer development and metastasis.

Project description:BackgroundThe oncoprotein c-Myc has been intensely studied in breast cancer and mouse mammary tumor models, but relatively little is known about the normal physiological role of c-Myc in the mammary gland. Here we investigated functions of c-Myc during mouse mammary gland development using a conditional knockout approach.ResultsGeneration of c-mycfl/fl mice carrying the mammary gland-specific WAPiCre transgene resulted in c-Myc loss in alveolar epithelial cells starting in mid-pregnancy. Three major phenotypes were observed in glands of mutant mice. First, c-Myc-deficient alveolar cells had a slower proliferative response at the start of pregnancy, causing a delay but not a block of alveolar development. Second, while milk composition was comparable between wild type and mutant animals, milk production was reduced in mutant glands, leading to slower pup weight-gain. Electron microscopy and polysome fractionation revealed a general decrease in translational efficiency. Furthermore, analysis of mRNA distribution along the polysome gradient demonstrated that this effect was specific for mRNAs whose protein products are involved in milk synthesis. Moreover, quantitative reverse transcription-polymerase chain reaction analysis revealed decreased levels of ribosomal RNAs and ribosomal protein-encoding mRNAs in mutant glands. Third, using the mammary transplantation technique to functionally identify alveolar progenitor cells, we observed that the mutant epithelium has a reduced ability to repopulate the gland when transplanted into NOD/SCID recipients.ConclusionWe have demonstrated that c-Myc plays multiple roles in the mouse mammary gland during pregnancy and lactation. c-Myc loss delayed, but did not block proliferation and differentiation in pregnancy. During lactation, lower levels of ribosomal RNAs and proteins were present and translation was generally decreased in mutant glands. Finally, the transplantation studies suggest a role for c-Myc in progenitor cell proliferation and/or survival.

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Dichloroacetate affects proliferation but not apoptosis in canine mammary cell lines.

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Dichloroacetate affects proliferation but not apoptosis in canine mammary cell lines.

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