Differentiating Agent Sodium Butyrate Research Articles

Nuclear factor of activated T cells (NFAT) signaling regulates PTEN expression and intestinal cell differentiation

The nuclear factor of activated T cell (NFAT) proteins are a family of transcription factors (NFATc1-c4) involved in the regulation of cell differentiation and adaptation. Previously we demonstrated that inhibition of phosphatidylinositol 3-kinase or overexpression of PTEN enhanced intestinal cell differentiation. Here we show that treatment of intestinal-derived cells with the differentiating agent sodium butyrate (NaBT) increased PTEN expression, NFAT binding activity, and NFAT mRNA expression, whereas pretreatment with the NFAT signaling inhibitor cyclosporine A (CsA) blocked NaBT-mediated PTEN induction. Moreover, knockdown of NFATc1 or NFATc4, but not NFATc2 or NFATc3, attenuated NaBT-induced PTEN expression. Knockdown of NFATc1 decreased PTEN expression and increased the phosphorylation levels of Akt and downstream targets Foxo1 and GSK-3α/β. Furthermore, overexpression of NFATc1 or the NFATc4 active mutant increased PTEN and p27(kip1) expression and decreased Akt phosphorylation. In addition, pretreatment with CsA blocked NaBT-mediated induction of intestinal alkaline phosphatase (IAP) activity and villin and p27(kip1) expression; knockdown of either NFATc1 or NFATc4 attenuated NaBT-induced IAP activity. We provide evidence showing that NFATc1 and NFATc4 are regulators of PTEN expression. Importantly, our results suggest that NFATc1 and NFATc4 regulation of intestinal cell differentiation may be through PTEN regulation.

Regulation of PTEN Expression in Intestinal Epithelial Cells by c-Jun NH2-Terminal Kinase Activation and Nuclear Factor-κB Inhibition

The tumor suppressor protein phosphatase and tensin homologue deleted on chromosome ten (PTEN) plays an important role in intestinal cell proliferation and differentiation and tumor suppression by antagonizing phosphatidylinositol 3-kinase. Despite its importance, the molecular mechanisms regulating PTEN expression are largely undefined. Here, we show that treatment of the colon cancer cell line HT29 with the differentiating agent sodium butyrate (NaBT) increased PTEN protein and mRNA expression and induced c-Jun NH2-terminal kinase (JNK) activation. Inhibition of JNK by chemical or genetic methods attenuated NaBT-induced PTEN expression. In addition, our findings showed a cross-talk between nuclear factor kappaB (NF-kappaB) and JNK with respect to PTEN regulation. Overexpression of the NF-kappaB superrepressor increased PTEN expression and JNK activity, whereas overexpression of the p65 NF-kappaB subunit reduced both basal and NaBT-mediated JNK activation and PTEN expression. Moreover, we showed that overexpression of PTEN or treatment with NaBT increased expression of the cyclin-dependent kinase inhibitor p27(kip1) in HT29 cells; this induction was attenuated by inhibition of PTEN or JNK expression or overexpression of p65. Finally, we show a role for PTEN in NaBT-mediated cell death and differentiation. Our findings suggest that the JNK/PTEN and NF-kappaB/PTEN pathways play a critical role in normal intestinal homeostasis and colon carcinogenesis.

Sodium butyrate-induced differentiation of human LIM2537 colon cancer cells decreases GSK-3beta activity and increases levels of both membrane-bound and Apc/axin/GSK-3beta complex-associated pools of beta-catenin.

Analysis of the glycogen synthase kinase-3beta (GSK-33) activity in several colon cancer cell lines suggested a correlation between comparatively low enzyme activity and moderate to high differentiation status. Treatment of LIM2537 cells, a poorly differentiated colon cancer cell line, with the potent differentiating agent sodium butyrate resulted in 34% reduction in GSK-3beta activity in the treated cells (P < 0.028, n = 3). Decreases in GSK-3beta activity were paralleled by stabilization of cytoplasmic beta-catenin, a hallmark of Wnt signaling. However, in contrast to Wnt signaling, expression of the beta-catenin/ TCF target genes c-myc and cyclin D1 did not appear to be increased in the sodium butyrate-treated cells. Interestingly, expression of membrane-bound beta-catenin was increased in the sodium butyrate-treated cells. This suggests that, in the context of cellular differentiation, increases in beta-catenin expression may be sequestered at the cell membrane and suggests that a possible role of sodium butyrate in promoting differentiation may be via increasing the levels of beta-catenin available for cell adhesion.

Vincristine- and cisplatin-induced apoptosis in human retinoblastoma. Potentiation by sodium butyrate

Chemotherapy alone has largely been unsuccessful in controlling retinoblastoma growth, and has traditionally been limited in use as an alternative to irradiation for the treatment of retinoblastoma. Recently, clinical studies combining chemotherapy with local therapies, including radiotherapy, laser therapy or cryotherapy and in some cases, cyclosporine A, have been effective in treating retinoblastoma. Differentiating agents may also be combined with chemotherapy to enhance the action of cytotoxic drugs on tumor cell growth, although this approach has not been fully investigated in retinoblastoma. In this study, we evaluated the cytotoxic response of human retinoblastoma cell lines (Y79 and WERI-Rb1) to two chemotherapy agents commonly used in treating retinoblastoma, vincristine (VCR) and cisplatin (CDDP). Retinoblastoma cells have been shown to be sensitive to the differentiating agent sodium butyrate, and cell lines were also treated with a combination of VCR or CDDP with sodium butyrate, and the effects on retinoblastoma viability assessed. Both VCR and CDDP induced dose-dependent death of Y79 and WERI-Rb1 cells, accompanied by nuclear and cytoplasmic condensation and DNA laddering, features characteristic of apoptosis. Inhibitors of macromolecular synthesis, cycloheximide and actinomycin-D, significantly reduced VCR- and CDDP-induced apoptosis, although putative endonuclease inhibitors zinc sulphate and aurintricarboxylic acid had no apparent effect. Treatment with 0.5 mM or 1 mM sodium butyrate combined with VCR or CDDP significantly increased induction of apoptosis by these agents. This augmentation of chemotherapy-induced apoptosis may have implications for retinoblastoma therapy.

Chronic ethanol promotes the neuronal differentiation of NG108-15 cells independently of toxin-sensitive G-proteins

The ability of ethanol to promote neuronal differentiation of NG108-15 mouse neuroblastoma x rat glioma hybrid cells was investigated using morphological, biochemical and electrophysiological techniques. Ethanol concentration—(10–200 mM) and time—(1 h–3 days) dependently reduced cell proliferation, but increased acetylcholinesterase (AChE) activity and cell protein content. Chronic ethanol (200 mM) also time-dependently increased voltage-sensitive Ca 2+ currents in the cells. Similar effects were obtained with chronic treatment of the cells with the standard differentiating agents sodium butyrate or forskolin. Chronic treatment of NG108-15 cells with primary alcohols (0.1–200 mM) of varying chain length all reduced cell proliferation and increased cell protein content and AChE activity with the potency order butanol>propanol>ethanol>methanol. Chronic treatment of NG108-15 cells with cholera toxin (50 ng ml −1) or pertussis toxin (50 ng ml −1) did not induce differentiation of the cells, nor did it modify the effects of 50 or 200 mM ethanol on cell proliferation, AChE activity or cellular protein content. Chronic cholera toxin did however abolish agonist-stimulated adenylyl cyclase activity in the cells, whereas pertussis toxin abolished receptor-mediated inhibition of adenylyl cyclase activity. Furthermore, inhibitors of protein kinase C (GF 109203X, 5 μM), protein kinase A (H-89, 10 μM) or Ca 2+/calmodulin-dependent protein kinase II (KN-62, 3 μM) all failed to modify the effects of 200 mM ethanol on cell proliferation, AChE activity and cellular protein content. These experiments indicate that chronic ethanol is able to promote neuronal differentiation of NG108-15 cells independently of toxin-sensitive G-proteins and some protein kinases.

Apoptosis induced by sodium butyrate treatment increases immunogenicity of a rat colon tumor cell line.

We have recently demonstrated that a treatment combining the cell differentiating agent sodium butyrate (NaBut) and interleukin-2 (IL2) resulted in a remission of established peritoneal colorectal carcinomatosis in rats. NaBut or IL2 treatment alone, never cured these tumour-bearing rats. In the present investigation, we report that NaBut-treatments induce apoptosis in the colonic cancer cells both in vitro and in vivo. We postulated that the significant therapeutic effect of NaBut/IL2 treatment can be mainly attributed to a NaBut-induced apoptosis of the tumoural cells increasing their immunogenicity. Indeed, treatment which combined apoptotic bodies (apobodies) as cell vaccine, plus IL2 immunotherapy significantly increased tumour remission and survival rate of the vaccinated rats, whereas IL2 treatment alone did not. We observed that the cured rats presented long-term protection against subsequent challenge with the parental tumour cells. This latter result suggests that these treatments generate an immune protection. This was confirmed by the presence, in the sera of the cured rats, of anti-tumoural antibodies directed against both the apobodies and the tumour cells, but not against normal colonocytes. In addition, we show that injections of apobodies before administration of the parental tumour cells results in a partial protection. We provide the first evidence that apobodies, derived from cancer cells after NaBut-treatment, induce a specific immune response against parental tumours cells. These data suggest that the distinctive immunologic properties of apobodies could provide a valuable tool in colorectal cancer immunotherapy.

Stable differentiation of a human colon adenocarcinoma cell line by sodium butyrate is associated with multidrug resistance

Colorectal cancers are often composed of cell types representing various differentiated cell lineages, however little is known concerning the relationship of differentiation and drug resistance in these cancers. The present study was performed to develop and characterize a stable, differentiated clone of the human colon cancer cell line LS174T and to characterize the drug resistance of this cell line in relation to its undifferentiated parental cell line. LS174T cell line was treated with the differentiating agent sodium butyrate (0.5 mM) for 30 days, then recultured in standard medium. Foci of flat-appearing cells appeared and were isolated using cloning rings, and subcloned. One subclone was designated LS174T-D. The LS174T-D clone maintains a stable, differentiated phenotype in standard culture conditions in the absence of sodium butyrate. It is characterized by the formation of a polarized monolayer with dome formation and the presence of prominent apical microvilli and tight junctions. This cell line demonstrated reduced growth in soft agar and nude mice compared with the parental cell line. LS174T-D cells expressed immunoreactive intestinal mucin antigens and brush border enzymes dipeptidyl aminopeptidase (DAP)-IV and aminopeptidase. The activities of DAP-IV and aminopeptidase were increased 5.6-fold and 3.4-fold, respectively, in LS174T-D compared with parental cells. Proliferation assays demonstrated that, compared with the parental cell line, LS174T-D cells were more resistant to doxorubicin (93-fold), cisplatin (23-fold), 5-fluorouracil (12-fold), 5-fluorodeoxyuridine (31-fold), and methotrexate (12.5-fold). Intracellular uptake of (3H)-5-fluorodeoxyuridine did not differ significantly in the differentiated and undifferentiated cell lines. Levels of mdr-1 p-glycoprotein measured by Western blot and RNA Northern blot assays were also similarly low in both cell lines. However, total glutathione content and glutathione-S-transferase activities were increased in LS174T-D cells by sixfold and threefold, respectively, compared with parental cells. Depletion of glutathione by pretreatment with DL-buthionine sulfoximine reversed LS174T-D resistance to cisplatin. Long-term treatment with sodium butyrate induces or selects for colon cancer cells with features of enterocytic differentiation. This stably differentiated cell line is associated with glutathione-mediated multidrug resistance, and provides a model for further studies of differentiation in normal and cancerous colon.

Sodium butyrate induces neuroendocrine cytodifferentiation in the insulinoma cell line RINm5F.

The differentiating agent sodium butyrate inhibits proliferation and stimulates cell-specific hormone expression in rat insulinoma cells. In this study, we investigated the effect of sodium butyrate on neuroendocrine cytodifferentiation in the rat insulinoma subclone, RINm5F. The cells were cultured with 0.5, 1, or 1.5 mM sodium butyrate for up to 72 h. Ultrastructurally, cells cultured with 1 mM sodium butyrate revealed a more differentiated appearance with an induction of cellular compartments involved in regulated insulin secretion. Morphometric analysis showed a significant elevation of neuroendocrine granule density. The total area of the specific granules was increased after incubation with 1 mM sodium butyrate for 48 and 72 h. Proliferation of RINm5F cells was inhibited by sodium butyrate in a dose-dependent manner. DNA production ceased completely within 24 h at 1.5 mM sodium butyrate. This concentration of sodium butyrate increased the cellular insulin content 8.9-fold and the insulin production 2-fold after 72 h. The insulin release was reduced from 79 +/- 3.5% in controls to 37 +/- 5.6% of total in a 24-h incubation period after 3 days of culture with 1.5 mM sodium butyrate. Insulin mRNA levels increased to a maximum of 324% compared with controls after 48 h of culture with 1.5 mM sodium butyrate. Chromogranin A mRNA levels increased to a similar extent (368 +/- 26%), whereas sodium butyrate did not stimulate the expression of synaptophysin, a major membrane component of small neuroendocrine vesicles. In conclusion, our data suggest the selective induction of neuroendocrine cytodifferentiation by sodium butyrate in RINm5F cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional regulation of prolactin receptor gene expression by sodium butyrate in MCF-7 human breast cancer cells.

The prolactin receptor (PRLR) mediates the diverse effects of prolactin, which in the mammary gland include the development of lobuloalveolar structures and increased tumor cell proliferation. Treatment of mammary carcinoma cells with the differentiating agent sodium butyrate (NaB) is known to reduce PRLR binding activity and PRLR gene expression, however the mechanism which mediates these changes is unknown, prompting this investigation. Using MCF-7 human breast cancer cells, assay of the rate of PRLR gene transcription by the nuclear run-on technique indicated that 3 mM NaB reduced PRLR gene transcription by 50% after 3 h of treatment and that this effect was maintained for at least 24 h. The protein synthesis inhibitor cycloheximide failed to abrogate this effect, which indicated that NaB did not require continuing protein synthesis to reduce the rate of PRLR transcription. Measurement of PRLR mRNA stability, using Northern blot analysis at various times after the inhibition of transcription with actinomycin D, showed that NaB treatment did not alter PRLR mRNA half-life. These results indicate that NaB inhibits PRLR gene expression by a transcriptional mechanism that does not require continuing protein synthesis.

Growth kinetics and chemoprevention of aberrant crypts in the rat colon.

Single and multiple colonic crypts exhibiting dysplasia that are detectable in situ by staining of rat colon with methylene blue are called aberrant crypts (AC) and may serve as an intermediate marker for colon cancer. In a characterization study, we have established the kinetics of AC growth and development over a period of 20 d following injection of rats with the carcinogen azoxymethane (AOM). AC are not present at 5 d post-injection, but are a constant feature at 10 d and thereafter. Multiple AC, presumably clonal, begin to evolve at 10 d and are consistent by 20 d, forming incipient microadenomata. We have examined 20 candidate chemopreventive agents for inhibition of AC. All agents were given in AIN-76 diet, at two dose levels, with injections of AOM. AC were measured after 5 weeks of growth. Among the most active AC-inhibiting agents were BHA, DFMO, quercetin, diallyl sulfide, 18 beta-glycyrrhetinic acid, and ascorbyl palmitate. In a post-initiation study, the differentiating agent sodium butyrate was ineffective, but piroxicam was highly effective in modulating AC growth. Further, piroxicam inhibited AC development at all stages of growth from single to polycryptal clusters of AC. The AC assay shows marked sensitivity and specificity for screening agents for chemoprevention of colon cancer.

Effect of dimethylsulfoxide on the proliferation and glycosaminoglycan synthesis of rat prostate adenocarcinoma cells (PAIII) in vitro: Isolation and characterization of DMSO‐resistant cells

Dimethylsulfoxide (DMSO) modulates the tumorigenicity and other characteristics of some malignant cell lines in vitro. We have investigated DMSO effects on cell proliferation and glycosaminoglycan (GAG) synthesis in rat prostate adenocarcinoma (PAIII) cells in culture. DMSO inhibited cell proliferation and GAG synthesis and shedding. Cells that survived the initial exposure to 2.5% DMSO could be propagated in this concentration of the agent and were designated PAIII-DMSO resistant (PAIII-DMSOr). PAIII-DMSOr cells showed reversible indications of increased differentiation such as decreased growth rate and saturation density. Although the PAIII-DMSOr cells were grown in 2.5% DMSO, they had normal or elevated GAG content. The major GAG of both PAIII and PAIII-DMSOr cells was undersulfated heparan sulfate. Verapamil, a calcium channel blocker that reverses drug resistance in tumor cells, stimulated the growth of PAIII-DMSOr cells in the presence of 2.5% DMSO, but was inhibitory in the absence of DMSO. Growth of PAIII cells was inhibited by the differentiating agents sodium butyrate and retinoic acid and by the ionophore monensin. Interestingly, growth of PAIII-DMSOr cells in the presence of 2.5% DMSO was largely unaffected by sodium butyrate or retinoic acid. The results suggest that (1) PAIII-DMSOr cells arise from the induction of a compensation mechanism to DMSO effects in a preexisting population of cells: (2) there is a correlation between GAG synthesis and cell proliferation; and (3) further study of the verapamil effect may help elucidate the mechanism of the DMSO-induced differentiation of cancer cells.

Modulation of the expression of a multidrug resistance gene (mdr-1/P-glycoprotein) by differentiating agents

Acquired resistance to multiple natural products in vitro is mediated by P-glycoprotein (Pgp). Expression of this protein has been demonstrated in some normal tissues and in tumor samples obtained from both untreated and treated patients. In situ hybridizations with RNA probes have demonstrated higher levels of expression of mdr-1/Pgp in well-differentiated tumors and in well-differentiated areas in tumors with mixed histologies. Expression of mdr-1/Pgp in human colon carcinoma cell lines was increased by the differentiating agents sodium butyrate, dimethyl sulfoxide, and dimethylformamide. In the SW-620 cell line addition of sodium butyrate resulted in a rapid induction of mdr-1/Pgp mRNA that was sustained for the duration of the exposure. The levels of P-glycoprotein were measured by immunoblotting and were also increased. Similar results were obtained in three other cell lines including the HCT-15 line. This induction occurred without alterations in nuclease sensitivity. Discontinuation of sodium butyrate was followed by a rapid fall in the levels of mRNA. The levels of P-glycoprotein returned to normal with a half-life of about 24 h. In spite of a 20-25-fold increase in the level of mdr-1/Pgp mRNA and P-glycoprotein, the SW-620 cell line did not demonstrate increased resistance to doxorubicin and vinblastine or decreased accumulation of vinblastine. In contrast, in the HCT-15 cell line, a 5-fold increase of mdr-1/Pgp was accompanied by a comparable fall in vinblastine accumulation which was reversed by verapamil. In the SW-620 cell line, the induced protein could be photolabeled using [3H]azidopine. Expression of mdr-1/Pgp appears to correlate with the degree of differentiation. However, its induction is not always accompanied by expression of the multidrug-resistance phenotype.

Effects of the Differentiating Agents Sodium Butyrate and N-Methylformamide on the Oxygen Enhancement Ratio of Human Colon Tumor Cells

We have previously shown that chronic adaptation of human tumor cells to the differentiation-inducing agents N-methylformamide (NMF) and sodium butyrate (NAB) increases the sensitivity of oxic cells to graded single doses of X rays. These studies were carried out to define the sensitivity of hypoxic cells after adaptation. Clone A colon tumor cells were grown for three passages in medium containing 170 mM NMF or 2 mM NAB and irradiated in suspension culture, after gassing with either oxygen (60 min) or ultrapure nitrogen (90 min), and complete survival curves were generated. Using the linear-quadratic equation to describe the data, it was found that NMF and NAB produced increased X-ray killing of hypoxic cells. At the 10% level of survival, the dose-modifying factors were about 1.20 and 1.25 for NMF- and NAB-adapted hypoxic cells, respectively, as compared to hypoxic control cells. However, since both oxic and hypoxic cells exhibited increased sensitivity after NMF and NAB adaptation, there was no major change in the oxygen enhancement ratio.

Contrasting effects of the differentiating agent sodium butyrate on recovery processes after x-irradiation in heterogeneous human colon tumor cells

The X ray survival responses of two clonal subpopulations of cells (clones A and D) from a heterogeneous human colon adenocarcinoma (DLD-1) were studied in unfed plateau phase cultures either as control cultures or in cultures grown for three passages in medium containing the differentiating agent sodium butyrate (NaB, 2 mM). Specifically, the cultures were studied with regard to their ability to express both potentially lethal and sublethal damage recovery (PLDR and SLDR). Growth in NaB-containing medium enhanced the radiation sensitivity of both cell lines in the low dose (“shoulder”) region of the survival curve. For clone A, the Dq value was reduced by 59%, and for clone D, the Dq value was reduced by 96%. NaB treatment increased both the rate and the extent of PLDR in both cell lines as assessed by single dose kinetic studies. However, when split dose experiments are performed to assess the expression of SLDR, NaB pretreatment was shown to totally inhibit the expression of SLDR, and also to alter the expression of PLDR under these conditions. These data suggest that PLDR and SLDR are separate, yet related, cellular recovery processes. In addition, NaB may be useful as an adjunct to radiotherapy by virtue of its ability to sensitize tumor cells in the dose range conventionally used for therapy, as well as by inhibition of sublethal damage recovery.