Charles desBordes

Induction of histone acetylation and inhibition of growth of mouse erythroleukemia cells by S-allylmercaptocysteine

Growth-inhibitory effects on DS19 mouse erythroleukemia cells were seen in the micromolar concentration range with allicin and S-allylmercaptocysteine and in the millimolar range with allyl butyrate, allyl phenyl sulfone, and S-allyl cysteine. Increased acetylation of histones was induced by incubation of cells with the allyl compounds at concentrations similar to those that resulted in the inhibition of cell proliferation. The induction of histone acetylation by S-allylmercaptocysteine was also observed in Caco-2 human colon cancer cells and T47D human breast cancer cells. In contrast to the effect on histone acetylation, there was a decrease in the incorporation of phosphate into histones when DS19 cells were incubated with 25 μM S-allylmercaptocysteine. Histone deacetylase activity was inhibited by allyl butyrate, but there was little or no effect with the allyl sulfur compounds examined in this study. A similar degree of downregulation of histone deacetylase and histone acetyltransferase was observed when DS19 cells were incubated with S-allylmercaptocysteine or allyl isothiocyanate. The induction of histone acetylation by S-allylmercaptocysteine was not blocked by a proteasome inhibitor. The mechanism by which S-allylmercaptocysteine induces histone acetylation remains to be characterized. It may be related in part to metabolism to allyl mercaptan, which is a more effective inhibitor of histone deacetylase.

Induction of histone acetylation in mouse erythroleukemia cells by some organosulfur compounds including allyl isothiocyanate

In previous studies we observed that some allyl sulfides can cause increased acetylation of histones and differentiation in DS19 mouse erythroleukemia cells. In the present work we observed increased acetylation of histones with allyl isothiocyanate and butanethiol but not with butyl sulfide or butyl disulfide. Increased acetylation of histones was established by change in electrophoretic mobility, incorporation of [3H]acetate or immunoblotting. Histone deacetylase in nuclei of DS19 cells was inhibited 74% by 0.5 mM allyl mercaptan and 43% by 0.5 mM butanethiol but was not significantly affected by 0.5 mM allyl isothiocyanate. There was some degree of reversibility in the effect of allyl isothiocyanate when the cells were incubated for 15 hr in fresh medium. The data suggested that allyl isothiocyanate may stimulate histone acetylation rather than inhibit histone deacetylation. Addition of allyl isothiocyanate, however, had very little or no additional effect on the induction of histone acetylation caused by trichostatin A. Histone acetyltransferase activity determined in cell homogenates was not increased by preincubation of cells with allyl isothiocyanate or inclusion of allyl isothiocyanate in the assay medium. It was concluded that treatment of mouse erythroleukemia cells with allyl isothiocyanate can cause increased acetylation of histones but the mechanism for this effect requires further elucidation.

Induction of histone acetylation and inhibition of growth by phenyl alkanoic acids and structurally related molecules.

PurposeA structure-activity study was undertaken to determine the influence of side chain length of phenyl alkanoic acids and the degree of unsaturation of phenyl alkenoic acids on the induction of histone acetylation and inhibition of cancer cell proliferation.Materials and methodsStudies on cell proliferation were performed with DS19 mouse erythroleukemic cells, PC-3 human prostate cancer cells and Caco-2 human colon cancer cells. Actions on histone deacetylase and the induction of histone acetylation were compared for 4-phenylbutyrate and structurally related molecules.ResultsIncreasing inhibition of cell proliferation by phenyl alkanoic acids together with a decrease in cells in S phase and an increase in apoptotic cells was observed with increased chain length between four and ten carbons. Introduction of double bonds into the side chain was associated with increased growth inhibition. In contrast, 4-phenylbutyrate was a more potent inhibitor of histone deacetylase and inducer of histone acetylation than the other phenyl alkanoic acids examined.ConclusionsIn comparison with the action of 4-phenylbutyrate, actions other than inhibition of histone deacetylase appear to be more important for growth inhibition by longer chain phenyl alkanoic and phenyl alkenoic acids.

Linoleic and linolelaidic acids differentially influence proliferation and apoptosis of MOLT-4 leukaemia cells.

The effects of varying concentrations of linoleic acid and its transisomer linolelaidic acid on the proliferation the ultrastructural morphology of MOLT‐4 T‐lymphoblastic leukaemia cells were investigated. At 2 and 4 days after exposure to the fatty acids, the cells were counted by flow cytometry and observed by electron microscopy. After 4 days of treatment, linoleic acid was growth stimulatory at concentrations of 200μm or less, but was markedly inhibitory at 400μm. In contrast, linolelaidic acid stimulated proliferation at concentrations of 100 and 200μm, but inhibited cell growth at 400μm. Cells treated with 400μm linoleic acid displayed dense accumulations of characteristic lipid globules and glycogen granules, and exhibited ultrastructural evidence of apoptosis including vacuolization, membrane blebbing and chromatin margination at the nuclear periphery. These results support the notion that geometrical isomerism and concentration of polyunsaturated fatty acids influence the proliferative destiny of cancer cells. Reverse transcription polymerase chain reaction (RT‐PCR) analysis revealed a previously documented larger alternatively spliced p53 gene transcript in MOLT‐4 cells cultured under reduced serum conditions. However, only wild‐type p53 transcripts were amplified by RT‐PCR of MOLT‐4 cells exposed to phytohaemagglutinin, linoleic acid or linolelaidic acid.

Growth inhibition of colon cancer cells by compounds affecting AMPK activity

AIMnTo determine if other molecules reported to modulate AMP-dependent protein kinase (AMPK) activity would have effects resembling those of metformin and phenformin on colon cancer cell proliferation and metabolism.nnnMETHODSnStudies were performed with four human colon cancer cell lines, Caco-2, HCT116, HT29 and SW1116. The compounds that were studied included A-769662, 5-aminoimidazole-4-carboxamide-1-ribofuranoside, butyrate, (-)-epigallocatechin gallate (EGCG), KU-55933, quercetin, resveratrol and salicylates. The parameters that were measured were cell proliferation and viability, glucose uptake, lactate production and acidification of the incubation medium.nnnRESULTSnInvestigations with several molecules that have been reported to be associated with AMPK activation (A-769662, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside, EGCG, KU-55933, quercetin, resveratrol and salicylates) or AMPK inhibition (compound C) failed to reveal increased medium acidification and increased glucose uptake in colon cancer cells as previously established with metformin and phenformin. The only exception was 5-aminosalicylic acid with which there were apparently lower glucose levels in the medium after incubation for 72 h. Further study in the absence of cells revealed that the effect was an artifact due to inhibition of the enzyme-linked glucose assay. The compounds were studied at concentrations that inhibited cell proliferation.nnnCONCLUSIONnIt was concluded that treatment with several agents that can affect AMPK activity resulted in the inhibition of the proliferation of colon cancer cells under conditions in which glucose metabolism is not enhanced, in contrast to the effect of biguanides.

Abstract 5439: Inhibition of growth and induction of differentiation of colon cancer cells by extracts from okra (Abelmoschus esculentus) and drumstick (Moringa oleifera)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnIn previous studies we have observed that several polyphenolic molecules can inhibit growth and induce the synthesis of differentiation markers in colon cancer cells. In the present work we have sought to determine if similar activity can be observed with methanolic and ethanolic extracts from okra and drumstick. In okra, phenolic molecules and antioxidant activity were associated primarily with the seeds, whereas in drumstick the seeds had lower concentrations of phenolics and antioxidant activity than skin, pulp and seed coat. In studies with okra extracts we have concentrated on the effects of seed extracts because they were generally most inhibitory for cell proliferation as judged by protein yield, thymidine incorporation into DNA and tetrazolium salt reduction. With extracts from drumstick the greatest inhibition of growth was generally with seed coat extracts but anomalous results were obtained in assays of tetrazolium salt reduction. Inhibition was greater at low concentrations but at high concentrations the apparent effect was reversed. Studies in the absence of cells showed that a component of the drumstick seed-coat extract reacted directly to reduce the tetrazolium salt. Comparison with other parts of the drumstick indicated that this reduction was much greater with the seed coat extract. Small effects on colon cancer cell differentiation have been seen with extracts of okra seed and with pulp and seed extract from drumstick as judged by induction of alkaline phosphatase activity. In the case of okra seed extracts this effect was additive with the action of butyrate. Okra and drumstick have had a role in traditional medicine and the present data are encouraging for further studies on potential cancer chemopreventive action.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5439. doi:1538-7445.AM2012-5439

Effects of Biguanides on Growth and Glycolysis of Bladder and Colon Cancer Cells

Background/ Aim: There is evidence that inhibitory effects of biguanides on oxidative phosphorylation require uptake of biguanides into the mitochondria. In this study the action of two biguanides that enter the mitochondria (buformin and phenformin) were compared with the action of two biguanides with poor uptake (phenyl biguanide and proguanil). Materials and Methods: Effects on growth, glucose uptake and medium acidification were studied with two human colon cancer cells and seven bladder cancer cell lines. Results: Growth inhibition was greatest with proguanil followed by phenformin, buformin and phenylbiguanide. In contrast, increased glucose uptake and acidification of the medium was observed with buformin and phenformin, with no change or less acidification of the medium with phenyl biguanide and proguanil. Conclusion: The effect of biguanides on glucose metabolism requires mitochondrial uptake while the mechanism for growth inhibition by biguanides remains to be defined.

Abstract A60: Inhibition of growth of bladder cancer cells by compounds that affect glucose metabolism

The tendency of cancer cells to have enhanced rates of glycolysis presents a target for combination therapy with agents that affect glucose metabolism. We have explored the action of three compounds on a panel of eight human bladder cancer cell lines showing a spectrum of growth rates. The compounds were an inhibitor of PFKFB3, namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), an inhibitor of pyruvate dehydrogenase kinase, dichloroacetate, and, paradoxically, phenformin. The biguanide phenformin increases glucose metabolism in some cancer cell lines but it can also be growth inhibitory. Effects on glucose metabolism and lactic acid production were monitored respectively by an enzyme-linked colorimetric assay for glucose in the medium and changes in the pH of the medium reflected by the absorbance of phenol red at 560 nm. Changes in these parameters were most marked for the rapidly growing T24 and UM-UC-3 bladder cancer cells and generally showed parallel changes with control and treated cells. We had previously established growth inhibitory effects of 10µM 3PO on the eight bladder cancer cell lines monitored by staining with sulforhodamine B. Inhibitory effects of 2.5 mM dichloracetate have been observed with additive inhibitory effects when cells were treated with combinations of 3PO and dichloracetate. Effects of phenformin as a single agent were studied at 10, 25 and 50µM. Concentration-related increases in medium acidification and glucose uptake were most consistently seen with T24 cells. The effects with 50µM phenformin could be blocked by coincubation with 2.5 mM dichloracetate. On the other hand, additive inhibitory effects on growth may be observed with combinations of phenformin and dichloroacetate. Sensitivity to the actions of phenformin could not be simply related to growth rate. The T24 and UM-UC-3 cells have similar growth rates and had similar inhibitory effects on growth when treated with phenformin. However, medium acidification and glucose uptake were less affected in the UM-UC-3 cells. Although there are conditions in which phenformin appears to increase the Warburg effect as it relates to glucose metabolism, there can be additive growth inhibitory effects when bladder cancer cells are treated with phenformin in combination with agents that inhibit glucose metabolism. Citation Format: Michael A. Lea, Mansour Altayyar, Charles desBordes. Inhibition of growth of bladder cancer cells by compounds that affect glucose metabolism. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A60.

Abstract 32: Inhibition of cancer cell growth by combined treatment with lactate dehydrogenase (LDHA) inhibitors and either phenformin or inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3)

The tendency for enhanced glycolysis in cancer cells presents a target for chemotherapy. In previous studies we observed that proliferation of colon and bladder cancer cells can be inhibited by treatment with either phenformin or an inhibitor of PFKFB3 namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). In the present work we have examined the action of two inhibitors that are effective at lower concentrations than 3PO, namely 1-(3-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PQP) and 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15). The LDHA inhibitors that we chose to study in increasing order of IC50s were methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2) Citation Format: Michael A. Lea, Yolanda Guzman, Charles desBordes. Inhibition of cancer cell growth by combined treatment with lactate dehydrogenase (LDHA) inhibitors and either phenformin or inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 32.

Abstract 3363: Inhibition of glycolysis and proliferation of colon cancer cells by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) an inhibitor of 6-phosphofructo-2-kinase (PFKFB3)

In human colon cancer cells, glycolytic activity shows a positive correlation with proliferation. PFKFB3 is an important enzyme regulating glycolysis in many tumor cells. We have studied the action of an established inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) as a single agent and in combination with other molecules that affect glycolysis and proliferation in four colon cancer cell lines (Caco-2, HCT116, HT29 and SW1116). Glucose uptake and cell proliferation were inhibited by 3PO and butyrate and additive effects were seen with 10 µM 3PO and 1 mM butyrate. Induction of alkaline phosphatase activity serves as a marker of differentiation in colon cancer cells. The large induction of alkaline phosphatase activity by butyrate seen in most colon cancer cell lines was not observed with 3PO. Similar contrasting effects on induction of alkaline phosphatase activity were seen with rat Morris hepatoma 7777 cells where 2-3 fold induction was seen with 1 µM vorinostat or 1 mM butyrate but no significant effect was seen with 3PO in the range of 5-30 µM. The biguanides, metformin and phenformin, increase glycolysis but inhibit proliferation of colon cancer cells. After combination treatment with 3PO, glycolysis was inhibited, as judged by glucose uptake and acidification of the medium, and additive inhibitory effects on cell proliferation could be observed. For example, after a 72 hour incubation of Caco-2 cells with 10 µM 3PO, 25 µM phenformin or 1 mM metformin, as single agents, proliferation relative to controls was decreased 20%, 17% and 34%, respectively. For combinations of 3PO and phenformin or 3PO and metformin the decreases relative to controls were 48% and 58%, respectively. In conclusion, 3PO was an inhibitor of proliferation of colon cancer cells, especially the rapidly growing HCT116 and HT29 cell lines. 3PO may be a useful agent in combination with other drugs that inhibit colon cancer cell proliferation. Citation Format: Michael A. Lea, Raymond Geherty, Rachelle David, Charles desBordes. Inhibition of glycolysis and proliferation of colon cancer cells by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) an inhibitor of 6-phosphofructo-2-kinase (PFKFB3). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3363. doi:10.1158/1538-7445.AM2014-3363