Reciprocal regulation of α-fetoprotein and albumin gene expression by butyrate in human hepatoma cells

GABTBGENTEBGLGGY 1994;107:499-504

Reciprocal Regulation of a-Fetoprotein and Albumin Gene Expression by Butyrate in Human Hepatoma Cells TAKUYA TSUTSUMI,* AK10 IDO,* KAZUHIKO NAKAO,* KEISUKE HAMASAKI,* YUJI KATO,* AKIRA OHTSURU,+ KEISUKE NAKATA,* TAIKI TAMAOKI,§ and SHIGENOBU NAGATAKl* *The First Department of Internal Medicine and %epattment of Cell Pathology, Atomic Disease Institute, Nagasaki University School of Medicine, Nagasaki, Japan; and “Department of Medical Biochemistry, University of Calgary, Alberta, Canada

Backygrwnd/Aims: Butyrate, a product of colonic bacterial flora, functions as an antiprollferative agent and induces cell differentiation in a variety of cell types. In the present study, the effects of butyrate on cell growth and expression of ol-fetoprotein (AFP) and albumin genes in Hut+-7 human hepatoma cells were investigated. Methods: The HUH-~cells were treated with se dlum butyrate (O-l mmol/L), and numbers of viable cells were counted at 24,48, and 72 hours after treatment. To elucidate the effects of sodium butyrate on AFP and albumin gene expression, Northern blotting and transient chloramphenicol acetyltransferase plasmid transfection experiments were performed.Results: Cell growth was dose dependently inhibited by sodium butyrate. By Northern blot analysis, the level of AFP messenger RNAwas reduced by treatment with sodium butyrate, whereas the level of albumin messenger RNA was elevated by this treatment. In transient chloram phenicol acetyltransferase plasmidtransfection experiments, sodium butyrate repressed the AFP promoter activity but did not change the AFP enhancer or silencer activities. In contrast, the albumin promoter activity was stimulated by sodium butyrate. Conclusions: These results suggest that butyrate leads to the recip rocal differentiating regulation of AFP and albumin gene expression at the transcriptional level in human hepatoma cells.

growth factors and cytokines are involved in the regulation of both genes. In fact, epidermal growth factor suppresses the AFP enhancer activity, which possibly regulates both of the AFP and albumin genes, resulting in the

down-regulation

of

T

(AFP) and albumin genes are simi-

lar in structure and believed to be derived from a common ancestral gene.’ Both genes are arranged tandemly on human chromosome 4 and expressed at high levels during the fetal stage; after birth, AFP expression

decreases rapidly to an almost undetectable level, whereas albumin expression increases steadily.2,3 However, the AFP gene is reexpressed in hepatoma cells.4 Thus, the regulation of AFP and albumin gene expression seems to be closely associated with differentiation or malignant transformation of hepatocytes. There has been a great deal of progress in characterization of cis- and tram-acting elements regulating the AFP and albumin genes. Many

genes.5

Transforming

growth factor pl and hepatocyte growth factor repress AFP gene expression through the reduction of its promoter activity.6.7 In addition, colloid osmotic pressure reversibly regulates AFP and albumin gene expression through the modulation of their promoter activities.* Butyrate is a natural fermentation product of colonic bacterial flora9 and has varying effects on cell proliferation and differentiation

through alterations

in the chromo-

somal structure and gene expression.l-OY1’Although the regulatory mechanism

of gene expression by butyrate

is not fully understood, recent studies have shown that butyrate acts as an antiproliferative

agent through alter-

ing cell maturation in a variety of cancer cells.‘2-‘4 Clinical trials using sodium or arginine butyrate as inducers of cell differentiation

have been performed in patients

with hematopoietic malignancies and resulted in partial remission in some cases. 15.16 In the present study, the effects of sodium butyrate on cell growth and expression of AFP and albumin genes in HUH-7 human hepatoma cells were analyzed to elucidate its physiological

he a-fetoprotein

both

role in differentiation

of trans-

formed hepatocytes. Mater&W

and

MetiWs

Chemicals Sodium butyrate was purchased from Sigma Chemical Co. (St. Louis, MO). (a-32P7deoxycytidine triphosphate and Dthreo-~dichloroacetyl-l-14C] chloramphenicol were purchased Abbreviationsused/nthis paper AFP, a-fetopmteln; CAT, chloram phenicol ecetyltransferase; C/EBP, CCAAT/enhancer core binding protein. 0 1994 by the American Gastmentedogical Assocletlon 001B-BoB5/94/93.00

!500

TSUTSUMI ET AL.

GASTROENTEROLOGY Vol. 107, No. 2

from Amersham Japan (Tokyo, Japan). Lipofectin reagents were purchased from Gibco (Gaithersburg, MD). 0.1 mu

Cell Culture The HUH-7 cells were maintained in a chemically defined medium, IS-RPMI.”

OdrnY

Cell growth was analyzed using

l.OnY

24-well multiplates (Falcon Plastics, Los Angeles, CA), and 5 X lo4 cells were placed into each well and incubated at 37°C in 5% COz. Two days later, the medium was replaced with fresh medium or fresh medium containing 0.1,0.5,

or 1 .O

mmol/L of sodium butyrate. The cells were further incubated at 37’C in 5% COz, and numbers of viable cells were counted at 24,48,

and 72 hours after incubation using the trypan blue

dye exclusion method.

Northern Blot Analysis

Figure 1. Effect of sodium butyrate on cell growth. incubated with fresh IS-RPM1 or IS-RPM1 containing mmol/L sodium butyrate. Numbers of viable cells indicated times after incubation. The values are mean + SD (n = 6).

HUH-~ ceils are 0.1, 0.5, or 1.0 are counted at represented as

Total RNA was isolated from the cultured cells by the guanidium isothiocyanate method. Total RNA (10 pg) was fractionated on a 1% formaldehyde agarose gel, transferred to a nylon membrane, and hybridized with a E3*P]-labeled complementary DNA (cDNA) probe. AFP cDNA (pHAF-2),” albumin cDNA (palb-7),2 and human p-actin cDNA (Wake

Chemical Ltd., Osaka, Japan) were used as probes.

incubated with the fresh medium in the absence or presence of 0.1, 0.5, or 1.0 mmol/L sodium butyrate. Two days later, the cells were harvested and lysed by five cycles of freezing and thawing. The lysate was heated at 63°C for 10 minutes, and the supernatant was used for determination of the CAT activity as previously described.23 The amounts of proteins and

Chloramphenicol Acetyltransferase Plasmids

and pALO.3-CAT),

The chloramphenicol acetyltransferase (CAT) plasmids

pAFS.l[A2.7]-CAT,

incubation times were 50 kg and 180 minutes (pAL12-CAT

used in this study were previously described.19*20The pBR-

25 pg and 30 minutes (pAFS.l-CAT,

and PSV1.6(+31)-CAT), 15 minutes (pSVAF2.4-CAT and pSV2-CAT),

or 10 pg and respectively.

CAT plasmid contains the CAT coding sequence and the simian virus 40 polyadenylation signal but no upstream regulatory sequences. pAF5.1 -CAT contains 5.1 -kilobase pairs of the AFP 5’-flanking sequence linked to the CAT gene in pBR-CAT. PSVAF2.4-CAT region (-5.3

contains the 2.4-kilobase full AFP enhancer

to -2.9

kilobase) inserted at the 5’ end of the

CAT gene in pSVl’-CAT,

which contains the simian virus 40

TATA box but lacks most of the 72-base pair repeat sequence. [email protected]]-CAT

contains both the 2.4-kilobase full AFP

enhancer and 169-base kilobase (-2.9

pair promoter region but lacks 2.7-

kilobase to -170

base pair) fragment where

the AFP “silencer” region is identified. PSV1.6(+31)-CAT

is

constructed by insertion of the 31-base

to

pair (from -1790

-1760) core sequence of AFP major distal silencer at BglII site within the SV40 enhancer in PSV1.6-CAT.” PALlZ-CAT and pALO.3-CAT contain the 12-kilobase and 288-base

pair

fragment of the human albumin 5’-flanking sequence linked to the CAT gene in pBR-CAT.

Cell Transfection and CAT Assay Transfection was performed using 3 pg (pSVAF2.4CAT and pSV2-CAT), 5 pg (pAF5.1-CAT, [email protected]]CAT, and PSV1.6(+31)-CAT), or 10 pg (pAL12-CAT and pALO.3-CAT) of the plasmid DNA per dish (60 cm*) by the lipofection method.22 After transfection, HUH-7 cells were

Inhibition of Cell Growth by Sodium Butyrate Numbers of viable cells were counted at 24, 48, and 72 hours after incubation in the absence or presence of various concentrations (0.1, 0.5, or 1.0 mmol/L) of sodium butyrate.

Cell growth was significantly sup-

pressed by sodium butyrate in a dose-dependent manner (Figure 1).

Decrease in AFP Messenger RNA Level but Increase in Albumin Messenger RNA Level by Sodium Butyrate Total cellular RNA was extracted from the cells incubated for 48 hours with fresh media or fresh media containing various concentrations (0.1,0.5, or 1 .O mm011 L) of sodium butyrate. The levels of AFP messenger RNA (mRNA) were dose dependently reduced; in contrast, the levels of albumin mRNA were elevated by treatment with sodium butyrate. The levels of p-actin mRNA showed no significant changes by this treatment (Figure

AFP AND ALBUMIN REGULATION BY BUTYRATE

August 1994

A

6

C

AFP

albumin

The liver tissue receives the dual blood supply from the portal vein and hepatic artery. Although the

pactin

hepatic artery provides 4O%-60% 2kb-b

501

2kb-B

A

2kb-b

pAF 5.1-CAT

1234

1234

of the oxygen supply

pALlP.CAT

1234

Figure 2. Effects of sodium butyrate on the levels of APP, albumin, and pactin mRNA. Total RNA (10 pg) was hybridized with (A) [32Pl_ labeled AFP, (6) albumin, or (C) human g-actin cDNA probe as de scribed in Materials and Methods. Arrows indicate positions of AFP, albumin, and pactin mRNA. Lane 1. control; lane 2. 0.1 mmol/L of sodium butyrate; lane 3, 0.5 mmol/L of sodium butyrate;and lane 4, 1.0 mmol/L of sodium butyrate.

c 1

2

3

4

B 2). By the densitometric analysis in three separate experiments, 1 mmol/L sodium butyrate suppressed the AFP mRNA level by 60.4%

+ 8.2% (mean ? SD) and up-

regulated the albumin mRNA level by 52.1%

pAF5.1[~2.7] +

pSV1.6(+31] +

pSVAF2.4 +

-

psv2 +

pALO. n +

IfI 13.9%

(mean 2 SD).

Reciprocal Changes in AFP and Albumin Promoter Activities by Sodium Butyrate In an attempt to analyze the regulatory mechanism of AFP and albumin gene expression by sodium butyrate,

transient

CAT

plasmid

transfection

ments were performed. As shown in Figure pression from pAF5. l-CAT pressed

by sodium

pAF5.1A[2.73-CAT,

butyrate.

CAT

expression

from

pair promoter

region but lacks

kilobase to -170

100

iz 0

base pair)

E

fragment containing the AFP “silencer” region, was also suppressed by sodium butyrate. CAT activities from

,o

pAF5.1 -CAT and pAF5.1 A[2.7]-CAT were suppressed by approximately 70%, respectively, by 1 mmol/L so-

8

dium

(from -2.9

C

was dose dependently sup-

which contains the AFP full en-

hancer and 169-base 2.7-kilobase

experi-

3, CAT ex-

butyrate.

CAT expression from PSV 1.6( + 3 l)CAT, which contains the core sequence of the AFP silencer region, was not changed by treatment with sodium butyrate. CAT expression from pSVAF2.4-CAT con-

taining only the AFP full enhancer region or CAT expression from pSV2-CAT used as a control plasmid were not affected by this treatment. In contrast, CAT expression from pAL12-CAT, which has the full 5’-upstream regulatory region of the albumin gene, was dose dependently stimulated by sodium butyrate. CAT expression from pAL0.3-CAT, which contains only the albumin promoter region, was also elevated by treatment with 1 mmol/L sodium butyrate.

;50

I.lr T

I!! 8

0 ~An.1

pAyd$

PS;~,,

pSVAF2.4

psv2

pAL12

pALO.

Plgure 3. Effects of sodium butyrate on AFP- and albumin-CAT expression. (A) After transfection with pAF5.1CAT plasmid or pAL12-CAT plasmid, HUH-~ cells were further incubated for 49 hours with fresh media or fresh media containing indicated concentrations of sodium butyrate. Lane 1, control; lane 2, 0.1 mmol/L of sodium butyrate; lane 3, 0.5 mmol/L of sodium butyrate; and lane 4, 1.0 mmol/L of sodium butyrate. (B) After transfection with indicated plasmids, HuH7 cells were incubated in the absence or presence of 1 mmol/L sodium butyrate. CAT activities were assayed as described in Materials and Methods. ~-AC, 3acetylchloramphenicol; ~-AC, l-acetylchloramphenicol; Cm, chloramphenicol. (C) Quantitative analysis by densitometric scanning. CAT activities were expressed as mean 2 SD of acetylation (percentage) in three separate experiments. 0, control; ?? . 1 mmol/L sodium butyrate.

502

TSUTSUMI

ET AL.

GASTROENTEROLOGY

Vol. 107.

No. 2

to the liver, three fourths of the total hepatic blood sup-

leukemia resulted in an elimination of myeloblasts from

ply are provided by the portal vein.24 Deprivation of the

the peripheral blood, an increase in mature myeloid cells,

portal blood flow results not only in liver atrophy but also

and a reduction of bone marrow myeloblasts. Accord-

deteriorated functions on hepatocytes.25-27 In addition,

ingly, our results also suggest that butyrate may act

several groups have shown that mouse hepatocytes mi-

clinically as a pharmacological inducer of differentiation

grate to the liver and function indefinitely after intra-

in human hepatoma cells.

splenic transplantation.28,29 The portal venous blood

Recent studies have shown that patterns of regulation

contains various biologically active peptides; however,

of AFP and albumin involve the interaction of specific

butyrate is one of the key factors involved in the mainte-

trans-acting

nance of physiological functions of hepatocytes because

within their promoter regions.38 Sequence analysis of the

butyrate allows induction of cell differentiation in a variety of cell types12-14 and because its concentration in the

promoter

portal venous blood seems to be much higher than in the

patocyte nuclear factor 1. Papaconstantinou et al.39 indi-

hepatic or peripheral venous blood. In fact, Cummings

cated that the developmental regulation of AFP and albu-

factors with &acting fragments

sequences located

shows consensus

sequence

for

CCAAT/enhancer core binding protein (C/EBP) and he-

et a1.30 reported that the portal blood concentrations of

min was strongly associated with the combined activity

butyrate in humans ranged from 14 to 64 pmol/L and

of C/EBP and hepatocyte nuclear factor 1. In fact, both

were approximately eight times higher than the systemic

UEBP

blood concentrations. In the physiological condition, the

scription factors for the adult albumin gene, but they

metabolic degradation of butyrate is very rapid with a

cannot mediate transcription of the AFP gene.38 Because

half-life of approximately 6 minutes.16 However, hepato-

the regulation of hemoglobin switching induced by buty-

cytes are steadily exposed to a relatively high concentra-

rate is connected with an enhanced C/EBP activity in the human y-globin gene promoter,40 butyrate leads to

tion of butyrate through the portal blood flow. Further-

and hepatocyte nuclear factor 1 are positive tran-

more, the pharmacokinetic study showed that butyrate

the reciprocal regulation of AFP and albumin gene prob-

was accumulated selectively in the liver when injected intravenously in mice. 31 Although the dose of butyrate

ably through the altered interaction of UEBP

used in this study (O.l- 1.0 mmol/L) is higher than its

promoter regions.

corresponding c&acting

with the

elements located within their

physiological concentration in the portal blood, it does

Small and well-differentiated hepatomas as well as nor-

not appear to be widely different from the physiological

mal liver tissue receive the mixed blood supply from

condition of the liver. In the present study, sodium butyrate inhibited cell growth dose dependently in HUH-7 cells. The levels of AFP mRNA were reduced dose dependently by treatment with sodium butyrate. In contrast, the levels of albumin mRNA were up-regulated by this treatment. In

both the portal vein and hepatic artery!l

In accordance

with tumor progression, hepatomas are perfused exclusively from the hepatic arterial flow in part because of interruption of the portal venous flow with tumor thrombosis or compression of the portal branches by the tumor itself.41’42 Clinical observations also indicate that in-

transient CAT plasmid transfection experiments, sodium

creased AFP secretion by hepatoma cells is associated

butyrate repressed the AFP promoter activity, whereas

with tumor progression. 33V43 Based on these observations,

the albumin promoter activity was stimulated by sodium

it is possible that butyrate functions physiologically

butyrate. These results are in agreement with the previ-

through the portal blood flow to regulate the AFP and

ous findings that sodium butyrate suppressed AFP secre-

albumin gene expression in human hepatoma cells.

tion but stimulated albumin synthesis in PLC/PRF/5

With the recent advances in diagnostic and therapeutic modalities, the prognosis of patients with hepatoma has been much improved.42*44345 However, even then, surgical candidates were few because of widespread intrahepatic involvement or lack of hepatic reserve resulting from coexisting advanced cirrhosis.42*46 Butyrate has a low order of toxicity.‘5,‘6.32 Patients with leukemia or P-hemoglobinopathies have been treated with sodium butyrate as an agent altering cell maturation with no The clinical efficacy of this treatmajor side effects.15*16347

hepatoma cells.32 Several lines of evidence show that AFP and albumin secretions by hepatoma cells are not linked to the rate of cell proliferation but rather to the degree Thus, our results suggest that of cell differentiation. 33V34 sodium butyrate is a possible agent inducing hepatoma cells to have some of the differentiating properties that are only found in normal hepatocytes. Several investigators have encompassed the potential clinical relevance of sodium butyrate as a differentiation inducer.35-37 Novogrodsky et al.‘> showed that the parenteral administration of butyrate in a patient with acute myelogenous

ment has not yet been sufficient because of rapid metabolic degradation of sodium butyrate.32 However, the

AFT’AND ALBUMIN

August 1994

development of novel prodrug derivatives that can impart better pharmacokinetic qualities to butyric acid is in progress.48 Because the prognosis of patients with advanced or multinodular types of hepatoma remains poor, treatment with these derivatives in combination with the recent treatment modalities should be considered to

17.

18.

achieve more favorable prognosis of these patients. 19.

R-

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503

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Received December 7,1993. Accepted April 29,1994. Address requests for reprlnts to: Shlgenobu Nagataki, M.D., The Flrst Department of Internal Medicine, Nagasaki University School of Medicine, 7-l Sakamoto-machi, Nagasaki 852, Japan. Fax: (81) 958430255. The authors thank Kaori Yokoyama and Masako Matsuo for secre tarial and technical help.