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.

Inhibition of Growth by Combined Treatment with Inhibitors of Lactate Dehydrogenase and either Phenformin or Inhibitors of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3.

Enhanced glycolysis in cancer cells presents a target for chemotherapy. Previous studies have indicated that proliferation of cancer cells can be inhibited by treatment with phenformin and with an inhibitor of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB) namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). In the present work, 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) were investigated. The inhibitors of lactate dehydrogenase (LDHA) studied in order of half-maximal inhibitory concentrations were methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2) < isosafrole < oxamate. In colonic and bladder cancer cells, additive growth inhibitory effects were seen with the LDHA inhibitors, of which NHI-2 was effective at the lowest concentrations. Growth inhibition was generally greater with PFK15 than with PQP. The increased acidification of the culture medium and glucose uptake caused by phenformin was blocked by combined treatment with PFKFB3 or LDHA inhibitors. The results suggest that combined treatment with phenformin and inhibitors of glycolysis can cause additive inhibition of cell proliferation and may mitigate lactic acidosis caused by phenformin when used as a single agent.

Inhibition of Growth of Bladder Cancer Cells by 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one in Combination with Other Compounds Affecting Glucose Metabolism.

In seven out of eight human bladder cell lines that were examined herein, growth was more dependent on the presence in the incubation medium of glucose rather than glutamine. The exception was the slowly growing RT4 cells that were more glutamine-dependent. Growth of all the cell lines was reduced by an inhibitor of 6-phosphofructo-2-kinase/2,6-bisphosphatase 3, namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). Growth was also reduced by three compounds that reduce the conversion of glucose to lactate: namely 2-deoxyglucose, butyrate and dichloroacetate. Additive effects were seen when these molecules were combined with 3PO. Treatment of bladder cancer cells with phenformin resulted in growth inhibition that was frequently accompanied by increased glucose uptake and acidification of the medium that was blocked by co-incubation with 3PO. The actions of 3PO suggest that inhibitors of PFKB3 merit further investigation in the treatment of bladder cancer and they may be useful agents in combination with other drugs that inhibit cancer cell proliferation.

Dysregulated metabolism contributes to oncogenesis.

Cancer is a disease characterized by unrestrained cellular proliferation. In order to sustain growth, cancer cells undergo a complex metabolic rearrangement characterized by changes in metabolic pathways involved in energy production and biosynthetic processes. The relevance of the metabolic transformation of cancer cells has been recently included in the updated version of the review "Hallmarks of Cancer", where dysregulation of cellular metabolism was included as an emerging hallmark. While several lines of evidence suggest that metabolic rewiring is orchestrated by the concerted action of oncogenes and tumor suppressor genes, in some circumstances altered metabolism can play a primary role in oncogenesis. Recently, mutations of cytosolic and mitochondrial enzymes involved in key metabolic pathways have been associated with hereditary and sporadic forms of cancer. Together, these results demonstrate that aberrant metabolism, once seen just as an epiphenomenon of oncogenic reprogramming, plays a key role in oncogenesis with the power to control both genetic and epigenetic events in cells. In this review, we discuss the relationship between metabolism and cancer, as part of a larger effort to identify a broad-spectrum of therapeutic approaches. We focus on major alterations in nutrient metabolism and the emerging link between metabolism and epigenetics. Finally, we discuss potential strategies to manipulate metabolism in cancer and tradeoffs that should be considered. More research on the suite of metabolic alterations in cancer holds the potential to discover novel approaches to treat it.

Flavonol regulation in tumor cells.

Flavonols comprise a group of flavonoid molecules that are widely distributes in fruits and vegetables. There is epidemiological data to suggest that consumption of flavonols can be accompanied by decreased cancer incidence. The anti-oxidant activity of flavonols may have an important role in preventing carcinogenesis. Therapeutic potential of flavonols is indicated by their growth inhibitory action accompanied by a decrease in several hallmarks of cancer such as resistance to apoptosis. Multiple mechanisms of action have been reported for the action of flavonols on cancer cells. Particular emphasis has been directed to inhibitory effects on several protein kinases and on the potential for prooxidant effects. The diversity of actions presents a problem in trying to elucidate primary and secondary effects but it may be a strength of the therapeutic potential of flavonols that it renders development of resistance more difficult for cancer cells. Cancer chemotherapy is usually characterized by the use of drug combinations. Some additive or synergistic combinations have been identified for flavonols and this is an area of ongoing investigation. As with other polyphenolic molecules there have been questions of cellular uptake and bioavailability. Several investigations have been and are being conducted to modify the structures of flavonols with the goal of increasing bioavailability. At present many investigators are sufficiently encouraged by past observations that they are responding to the challenge to optimize the dietary and therapeutic use of flavonols in cancer prevention and treatment.

Growth inhibition of colon cancer cells by compounds affecting AMPK activity.

AIM: To 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. METHODS: Studies 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. RESULTS: Investigations 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. CONCLUSION: It 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.

Regulation of the proliferation of colon cancer cells by compounds that affect glycolysis, including 3-bromopyruvate, 2-deoxyglucose and biguanides.

In previous studies performed by our group, we observed that 2-deoxyglucose blocked the acidification of the medium used for culture of colon cancer cells caused by incubation with biguanides and it had an additive inhibitory effect on growth. In the present work, we found that 3-bromopyruvate can also prevent the lowering of pH caused by biguanide treatment. 3-Bromopyruvate inhibited colonic cancer cell proliferation, but the effect was not always additive to that of biguanides and an additive effect was more notable in combined treatment with 3-bromopyruvate and 2-deoxyglucose. The induction of alkaline phosphatase activity by butyrate was not consistently affected by combination with other agents that modified glucose metabolism. The drug combinations that were examined inhibited proliferation of wild-type and p53-null cells and affected colonic cancer lines with different growth rates.

Addition of 2-deoxyglucose enhances growth inhibition but reverses acidification in colon cancer cells treated with phenformin.

A report that effects of butyrate on some cells may be mediated by activation of AMP-activated protein kinase (AMPK) prompted this study which examines if other AMPK activators can induce differentiation and inhibit proliferation of colon cancer cells in a manner similar to butyrate. Using induction of alkaline phosphatase as a marker, it was observed that compound C, an AMPK inhibitor, is able to reduce the differentiating effect of butyrate on SW1116 and Caco-2 colon cancer cells. Metformin was observed to be less effective than butyrate in the induction of alkaline phosphatase but was more effective as a growth inhibitor. Phenformin was found to be a more potent growth inhibitor than metformin and both compounds cause acidification of the medium when incubated with colon cancer cells. Combined incubation of 2-deoxyglucose with either of the biguanides prevented the acidification of the medium but enhanced the growth inhibitory effects.

Inhibition of growth and induction of alkaline phosphatase in colon cancer cells by flavonols and flavonol glycosides.

We observed previously that quercetin can increase the activity of the differentiation markers alkaline phosphatase and dipeptidyl peptidase in Caco-2 colon cancer cells. In the present work, we compared the effects of quercetin on cell proliferation and differentiation with the action of related flavonols and quercetin glycosides. Relative to the action of quercetin, effects on growth and enzyme activities did not always follow parallel trends but quercetin 3-glucoside was notably more potent in both respects while quercetin rutinoside was less active. Of the compounds examined, baicalein and myricetin caused the greatest production of hydrogen peroxide when incubated with the medium. Flavonols can have pro-oxidant effects, but our data suggested that this action was not the sole determinant of growth inhibitory or differentiating effects on Caco-2 cells. Our data indicated that effects of quercetin on colon cancer cell lines can be greatly affected by glycoside modification.

Recently identified and potential targets for colon cancer treatment.

The systemic therapy for colorectal cancer has advanced from essentially a single, partially effective agent, 5-fluorouracil, to a combination of cytotoxics and antibodies offering increased survival. In addition to damage of DNA through agents, such as oxaliplatin and irinotecan, and inhibition of DNA replication, a promising approach involves modifying the control of gene expression, including epigenetic control. Modulation of invasion and metastasis should become increasingly important. Inhibition of growth-factor signaling with small-molecule drugs and antibodies can be a part of this effort. Further progress in the control of gene expression in colon cancer may be achieved with miRNAs and RNA interference if technical problems can be overcome. A number of genetic changes in colorectal cancer progression have been identified and offer targets for future therapy.

Inhibition of growth and induction of differentiation markers by polyphenolic molecules and histone deacetylase inhibitors in colon cancer cells.

Previously we found that a fruit-derived polyphenol fraction caused an inhibition of proliferation and an induction of differentiation markers in Caco-2 human colon cancer cells. In the present work, we sought to determine if individual polyphenols would exert similar actions. Proliferation was inhibited by several polyphenolic molecules including gallic acid, ellagic acid, quercetin and resveratrol. In Caco-2 cells, growth inhibition was accompanied by increased specific activities of two differentiation markers, alkaline phosphatase and dipeptidyl peptidase, but not of aminopeptidase. Increased enzyme activities were not seen in HT29 and SW1116 colon cancer cells. In Caco-2 cells there were additive effects of butyrate or valproate and polyphenolic molecules. Histone acetylation was not greatly affected by the polyphenols. Cycloheximide inhibited protein synthesis in the 3 cell types examined but paradoxically, in Caco-2 cells it caused increased specific activities of alkaline phosphatase and dipeptidyl peptidase. Several plant polyphenols can inhibit the growth of colon cancer cells but increased specific activity of some differentiation markers seen in Caco-2 cells did not appear to be a general phenomenon in colon cancer cells.

Inhibition of growth and induction of differentiation of colon cancer cells by peach and plum phenolic compounds.

The action of extracts from anthocyanin-enriched plums and peaches on growth and differentiation was studied with human colon cancer cells. Growth inhibitory effects were observed in Caco-2, SW1116, HT29 and NCM460 cells. In Caco-2 cells but not in the other cells studied there was evidence for increased differentiation as judged by increased activity of alkaline phosphatase and dipeptidyl peptidase. A differentiating effect on Caco-2 cells was not seen with cyanidin or cyanidin-3-glucoside but the action of the fruit extracts was additive with the action of butyrate and with the MEK1/2 inhibitor U0126. Fractionation using C18 indicated activity resided within a fraction containing anthocyanins but further fractionation using LH-20 suggested that most of the activity was in a fraction containing polyphenols other than anthocyanins. It was concluded that several peach and plum phenolic molecules can influence growth and differentiation in human colon cancer cells.

Induction of differentiation of colon cancer cells by combined inhibition of kinases and histone deacetylase.

The MAP kinase pathway inhibitor U0126 in combination with butyrate promotes differentiation in some colon cancer cell lines. We examined several inhibitors of histone deacetylase (HDAC) in combination with U0126 and other protein kinase inhibitors to see if these effects are general properties of HDAC inhibitors or butyrate alone. Alkaline phosphatase and peptidase activities were examined as markers for cellular differentiation in the human colon cancer cell lines Caco-2 and HT29 and the minimally transformed NCM460. Several HDAC inhibitors caused greater increases of alkaline phosphatase in the cancer cells than in NCM460, in which butyrate was the only HDAC inhibitor that caused a consistent increase. Unlike the JNK and PKC inhibitors examined, the MEK 1/2 inhibitor U0126 induced alkaline phosphatase activity in Caco-2 as a single agent and caused additive effects with HDAC inhibitors. The PI-3 kinase inhibitor LY294002 had little effect alone but enhanced the response of most HDAC inhibitors as did the raf inhibitor GW5074. In addition to butyrate, several HDAC inhibitors can induce differentiation in colon cancer cells and the responses may be enhanced by U0126, GW5074 and LY294002.

Inhibition of cell proliferation by potential peroxisome proliferator-activated receptor (PPAR) gamma agonists and antagonists.

This study was initiated to determine if potential PPAR gamma antagonists could block the inhibition of cell proliferation caused by 4-phenylbutyrate. The action of 4-phenylbutyrate differed from other PPAR gamma ligands examined in that it induces histone acetylation. Proliferation of DS19 mouse erythroleukemia cells was inhibited by PPAR gamma agonists (4-phenylbutyrate, rosiglitazone, ciglitazone and GW1929) and by potential PPAR gamma antagonists: BADGE (Biphenol A diglycidyl ether), GW9662, PD068235 and diclofenac. Combined incubations tended to exhibit additive inhibitory effects. Potential PPAR gamma agonists and antagonists inhibited the incorporation of thymidine into DNA of human prostate (PC3), colon (Caco-2) and breast (T47D) cancer cells but also affected NIH3T3 cells that have little or no expression of PPAR gamma. Lipid accumulation in T47D cells was seen after incubation with 4-phenylbutyrate and both potential PPAR gamma agonists and antagonists. The extent to which the effects of 4-phenylbutyrate on cell proliferation are mediated through PPAR gamma or induction of histone acetylation remains an open question. We conclude that potential PPAR gamma antagonists may fail to reverse the growth inhibitory effect of PPAR gamma ligands and may themselves act as growth inhibitory agents.

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

PURPOSE: A 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 METHODS: Studies 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. RESULTS: Increasing 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. CONCLUSIONS: In 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.

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 microM 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.

Exposure to a deuterated analogue of phenylbutyrate retards S-phase progression in HT-29 colon cancer cells.

Differentiation agents that induce neoplastic cells to regain a normal phenotype and/or cause growth arrest without significantly affecting normal cells represent an attractive option for cancer treatment. Analogues of short chain fatty acids, such as phenylbutyrate (PB), have been studied as clinically relevant agents. In an attempt to improve its pharmacokinetic profile, structural modifications of PB and other fatty acids have been studied. We hypothesize that strategic isotopic modification of PB would result in a longer half-life and thus translate into a more potent differentiation agent for clinical use. Using a colon cancer model, we demonstrated that 2,2,3,3-tetradeuterated PB (D4PB) significantly increased induction of apoptosis and inhibition of cell proliferation as compared with PB and butyrate. Difference in potency could not be explained by the effect of D4PB on the expression of specific regulatory proteins of the apoptotic cascade or from the inhibitory effect of D4PB on histone deacetylase activity. Interestingly, exposure of HT-29 colon cancer cells to D4PB resulted in a slowing of S transit, in contrast to butyrate and PB, which induced a G2/M cell cycle block. This difference in cell cycle effect may explain the differences seen in the potency of the phenotypic changes seen with treatment with D4PB. Further studies are needed to elucidate the mechanisms underlying effects of D4PB on the cell cycle.