dRLh-84 Cells Research Articles

Cardiac glycosides stimulate endocytosis of GLUT1 via intracellular Na+ ,K+ -ATPase α3-isoform in human cancer cells.

Glucose transporter GLUT1 plays a primary role in the glucose metabolism of cancer cells. Here, we found that cardiac glycosides (CGs) such as ouabain, oleandrin, and digoxin, which are Na+ ,K+ -ATPase inhibitors, decreased the GLUT1 expression in the plasma membrane of human cancer cells (liver cancer HepG2, colon cancer HT-29, gastric cancer MKN45, and oral cancer KB cells). The effective concentration of ouabain was lower than that for inhibiting the activity of Na+ ,K+ -ATPase α1-isoform (α1NaK) in the plasma membrane. The CGs also inhibited [3 H]2-deoxy- d-glucose uptake, lactate secretion, and proliferation of the cancer cells. In intracellular vesicles of human cancer cells, Na+ ,K+ -ATPase α3-isoform (α3NaK) is abnormally expressed. Here, a low concentration of ouabain inhibited the activity of α3NaK. Knockdown of α3NaK significantly inhibited the ouabain-decreased GLUT1 expression in HepG2 cells, while the α1NaK knockdown did not. Consistent with the results in human cancer cells, CGs had no effect on GLUT1 expression in rat liver cancer dRLh-84 cells where α3NaK was not endogenously expressed. Interestingly, CGs decreased GLUT expression in the dRLh-84 cells exogenously expressing α3NaK. In HepG2 cells, α3NaK was found to be colocalized with TPC1, a Ca2+ -releasing channel activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The CGs-decreased GLUT1 expression was significantly inhibited by a Ca2+ chelator, a Ca2+ -ATPase inhibitor, and a NAADP antagonist. The GLUT1 decrease was also attenuated by inhibitors of dynamin and phosphatidylinositol-3 kinases (PI3Ks). In conclusion, the binding of CGs to intracellular α3NaK elicits the NAADP-mediated Ca2+ mobilization followed by the dynamin-dependent GLUT1 endocytosis in human cancer cells.

Cytotoxic Effects of Hydroxylated Fullerenes in Three Types of Liver Cells.

Fullerenes C60 have attracted considerable attention in the biomedical field due to their interesting properties. Although there has been a concern that C60 could be metabolized to hydroxylated fullerenes (C60(OH)x) in vivo, there is little information on the effect of hydroxylated C60 on liver cells. In the present study, we evaluated the cytotoxic effects of fullerene C60 and various hydroxylated C60 derivatives, C60(OH)2, C60(OH)6–12, C60(OH)12 and C60(OH)36, with three different types of liver cells, dRLh-84, HepG2 and primary cultured rat hepatocytes. C60, C60(OH)2 and C60(OH)36 exhibited little or no cytotoxicity in all of the cell types, while C60(OH)6–12 and C60(OH)12 induced cytotoxic effects in dRLh-84 cells, accompanied by the appearance of numerous vacuoles around the nucleus. Moreover, mitochondrial activity in liver cells was significantly inhibited by C60(OH)6–12 and C60(OH)12. These results indicate that the number of hydroxyl groups on C60(OH)x contribute to the difference of their cytotoxic potential and mitochondrial damage in liver cells.

Possible Roles of the AP-1 Site in the Cytosolic T3 Binding Protein Promoter and Insights into its Physiological Significance

Cytosolic 3,5,3'-triiodo-l-thyronine-binding protein plays pivotal roles in the regulation of intracellular 3,5,3'-triiodo-l-thyronine concentration in vivo. The expression of the protein, which is identical to μ-crystallin, is regulated by various factors. To elucidate the mechanisms of its expression, we evaluated the promoter transactivity and insulin signaling via the AP-1 site in the promoter. The isolated 600 bp human and 1976 bp mouse 5'-flanking regions were cloned in a luciferase reporter plasmid. The luciferase activity was estimated in GH3, dRLh-84, HEK293, and insulin receptor-overexpressing CHO-IR cells. The effects of 12-O-tetradecanoylphorbol 13-acetate and insulin on μ-crystallin mRNA expression were evaluated in various cells. The region between -200 and the transcriptional start site was crucial for constitutive expression in μ-crystallin-expressing dRLh-84 cells. This region contained an AP-1 site. 12-O-Tetradecanoylphorbol 13-acetate increased the level of μ-crystallin mRNA expression in HEK 293 cells. The compound also increased luciferase activity through the promoter. Mutation in the AP1 site diminished the response to the compound. The promoter was also activated by insulin treatment in CHO-IR cells. Insulin treatment increased μ-crystallin mRNA expression in Raw264.7 cells, but decreased in HEK293, P19, and dRLH-84 cells. The expression of μ-crystallin was regulated through the AP-1 site in the promoter. The signals related to AP-1 activation, such as insulin signaling may have diverse effects on μ-crystallin mRNA expression.

Enhancement of cytotoxic and pro-apoptotic effects of 2-aminophenoxazine-3-one on the rat hepatocellular carcinoma cell line dRLh-84, the human hepatocellular carcinoma cell line HepG2, and the rat normal hepatocellular cell line RLN-10 in combination with 2-deoxy-D-glucose

The cytotoxic and pro-apoptotic effects of a single dose of 2-aminophenoxazine-3-one (Phx-3) or 2-deoxyglucose (2-DG) or of a combined dose of Phx-3 and 2-DG were studied in the rat hepatocellular carcinoma cell line dRLh-84, the human hepatocellular carcinoma cell line HepG2 and the rat normal hepatocellular cell line RLN-10. The number of viable cells decreased in a dose-dependent manner, when dRLh-84, HepG2 or RLN-10 cells were treated with 2-DG (0.5-20mM) or Phx-3 (1-50µM) alone at 37˚C for 48h. When these cells were treated with 10mM 2-DG and different concentrations of Phx-3, the number of viable cells decreased dose-dependently and in an additive manner for these agents. A single dose of 2 or 10µM Phx-3 induced apoptotic morphology characterized by nuclear condensation and cell shrinkage in dRLh-84, HepG2 and RLN-10 cells, while a single dose of 10mM 2-DG did not. When Phx-3 (2 or 10µM) treatment was combined with 2-DG (10mM) treatment in these three cell lines, the cells with apoptotic morphology increased extensively, which was confirmed by flow cytometric analysis. In addition, autophagic morphology characterized by cytosolic vacuole formation was significantly increased in the hepatocellular carcinoma cell lines dRLh-84 and HepG2 but not in the normal hepatocellular cell line RLN-10 after a single dose of Phx-3 or 2-DG or a combined dose of Phx-3 and 2-DG. Furthermore, when dRLh-84 and HepG2 cells were treated with Phx-3 alone or a combined dose of Phx-3 and 2-DG, depolarization of the mitochondria was extensive, but that of the normal cell line RLN-10 was not. These results may imply that the mechanism for the apoptosis of hepatocellular carcinoma cells caused by Phx-3 alone or a combined dose of Phx-3 and 2-DG differs from that of the normal cell line RLN-10. The present results demonstrate that Phx-3 alone may be beneficial for targeting liver cancer and that its anticancer activity may be enhanced by 2-DG. However, a combined dose of Phx-3 and 2-DG may exert adverse effects on normal liver cells, as evidenced by the cytotoxic and pro-apoptotic effects of the combined treatment in the rat normal hepatocellular cell line RLN-10.

Selective Regulation of Multidrug Resistance Protein in Vascular Smooth Muscle Cells by the Isoquinoline Alkaloid Coptisine

When the biological activites of hydrophobic drugs or xenobiotics are studied, it is important to clarify their effects on expression and function of multidrug resistance (MDR) protein. We therefore evaluated the effects of coptisine on MDR in comparison with the structurally related isoquinoline alkaloids berberine and palmatine. To achieve this, we investigated the effects of the three alkaloids on the expression and function of P-glycoprotein/MDR1, MDR1 gene products, in vascular smooth muscle cells (VSMCs). In A10 cells (a rat VSMC line), coptisine upregulated the mRNAs of Mdr1a and Mdr1b, rodent homologues of human MDR1, and these effects were completely abrogated by actinomycin D. Coptisine also induced Mdr1a/1b protein expression and enhanced the efflux of rhodamine 123 from A10 cells. In contrast, berberine and palmatine slightly upregulated the mRNAs of Mdr1a and Mdr1b, but failed to induce Mdr1a/1b protein expression or stimulate rhodamine 123 efflux. To clarify whether these effects occurred in other cells, the effects of the three alkaloids on Mdr1a/1b function were examined in 3Y1, dRLh-84 and B16 cells. Coptisine and berberine enhanced rhodamine 123 efflux in all three cell types, while palmatine inhibited it, based on the finding that palmatine efficiently activated the Mdr1a ATPase activity as a good substrate for Mdr1a. Therefore, the three isoquinoline alkaloids regulated MDR differently in cell type-specific manners. In particular, only coptisine induced Mdr1a/1b in A10 cells and stimulated rhodamine 123 efflux. Taken together, coptisine appears to exert VSMC-selective effects on Mdr1a/1b induction in contrast to berberine and palmatine.

ZHX2 and ZHX3 repress cancer markers in normal hepatocytes

ZHX2 and ZHX3 are the members of the ZHX transcriptional repressor family. To investigate the regulatory role of the repressors in hepatocytes and their involvement in carcinogenesis, the expression levels of ZHX2 and ZHX3 mRNAs were examined. The dRLh-84 hepatoma cells considerably expressed cancer marker genes PKM and HK II that are expressed in developing fetal tissues and cancer cells but repressed in normal hepatocytes. In dRLh-84 cells, the expression levels of ZHX2 and ZHX3 were very low compared with rat hepatocytes. Upon the reporter gene analysis utilizing the promoter region of these genes, ZHX3 repressed the transcription of the reporter luciferase gene from both promoters while ZHX2 only repressed that from HK II promoter. The promoter activity of alpha-fetoprotein was also repressed by the expression of ZHX2 in HLE hepatoma cells in a dose-dependent manner. We concluded that ZHX2 and ZHX3 were involved in the transcriptional repression of the hepatocellular cacinoma markers in normal hepatocytes, suggesting that the failure of the ZHX2 and/or ZHX3 expression might be a critical factor in the hepatocellular carcinogenesis.

Novel tumor‐ablation device for liver tumors utilizing heat energy generated under an alternating magnetic field

We have developed a novel tumor-ablation device for liver tumors utilizing heat energy induced by magnesium ferrite (MgFe(2)O(4)) particles under an alternating magnetic field (AMF) produced by electric currents. This novel device can repeatedly heat liver tumors at lower temperature than usual heating devices, such as radiofrequency ablation therapy, with slight infliction of pain. This study assesses its heating effect on rat liver tumors as local therapy. The small needle was manufactured from MgFe(2)O(4) particles by sintering at 1100 degrees C. After a MgFe(2)O(4) needle was inserted into liver tumors comprising of dRLh-84 cells, the tumors were heated for 30 min under an AMF. We examined cellular activity by using nicotinamide adenine dinucleotide (NADH) diaphorase staining and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) staining, and evaluated the effect of suppressing tumor growth by sequentially comparing the tumor diameter with that of the control group. The mean temperature of the heated tumors was 60.2 +/- 1.8 degrees C. The tumor cells were constricted, and chromatin of nuclei had shrunk immediately after heating. The heat-injury area that contained the tumors was negative for NADH diaphorase activity. After 3 days, the tumor cells in the heat-injury area became positive for TUNEL staining, which detects cell death. At 7 days, the mean tumor diameters were significantly smaller in the heating group than in the control group (6.15 +/- 0.47 mm vs 16.89 +/- 2.69 mm; P < 0.05). This device, utilizing heat energy induced by ferromagnetic metal under an AMF, appears useful as local thermotherapy for human liver cancer.

Oleic acid prevents apoptotic cell death induced by trans10, cis12 isomer of conjugated linoleic acid via p38 MAP kinase dependent pathway

Our previous study indicated that oleic acid prevented apoptotic cell death induced by trans10, cis12 (t10, c12)-conjugated linoleic acid in rat hepatoma dRLh-84 cells. The intracellular mechanism of action oleic acid is still unknown. Here, we showed that p38 mitogen-activated protein kinase (MAPK) inhibition using its specific inhibitor SB203580 cancelled the ameliorative effect of oleic acid on the cytotoxicity of t10, c12-conjugated linoleic acid. In addition, SubG1 cell population analysis showed that p38 MAPK played an essential role in the prevention of apoptotic cell death by oleic acid. In fact, p38 phosphorylation level was upregulated in cells treated with oleic acid irrespective of t10, c12-conjugated linoleic acid stimulation. Interestingly, t10, c12-conjugated linoleic acid increased intracellular triglyceride accumulation. However, oleic acid completely inhibited this effect. These observations indicated the involvement of blockade of a p38 MAPK pathway in the ameliorative effect of oleic acid on apoptosis induced by t10, c12-conjugated linoleic acid.

A Quinone Isolated from the Nest of Vespa simillima and Its Growth-Inhibitory Effect on Rat Liver Cancer Cells

A biologically active quinone that exhibits a growth-inhibitory effect on rat liver cancer cells was isolated from the nest of a social wasp, Vespa simillima (Vespinae, Vespidae). The structure was unambiguously determined by NMR and synthetic experiments and identified as being 7,8-seco-para-ferruginone. The synthetic quinone showed potent growth-inhibitory effects on rat hepatoma cells (IC50 value=1.9 microM). Morphological analyses of dRLh84 cells using the synthetic compound were tested. Chromatin coagulation was induced by the synthetic compound at 31.6 muM.

Selective inhibition of vascular smooth muscle cell proliferation by coptisine isolated from Coptis rhizoma, one of the crude drugs composing Kampo medicines Unsei-in

Acceleration of vascular smooth muscle cell (VSMC) proliferation is closely linked to the pathogenesis of vascular diseases. We, therefore, focused on traditional Japanese herbal medicines (Kampo medicines) used to ameliorate the impairment of microcirculation or blood stasis and screened them for their ability to inhibit rat VSMC proliferation. Among them, Unsei-in was found to effectively suppress VSMC proliferation, and Coptis rhizome was the responsible constituent crude drug. The extract of Coptis rhizome inhibited VSMC proliferation with the GI(50) value of 4.4 microg/ml, which was much lower than those against the proliferation of 3Y1, dRLh-84, B16, and HeLa cells. The Coptis rhizome extract inhibited the progression of VSMC arrested at G(0)/G(1) phase from G(0)/G(1) to S phase, but not that of 3Y1 cells. Biological assay-guided fractionation revealed that an alkaloid of Coptis rhizome, coptisine, was the active ingredient in selectively preventing VSMC proliferation with GI(50) of 3.3 microM (1.2 microg/ml). When the structurally-related isoquinoline alkaloids of protoberberine class were studied for their inhibitory activities, berberine decreased the VSMC proliferation with GI(50) of 95.1 microM (35.4 microg/ml), about 30 times higher concentration than coptisine, while palmatine failed to show any activity. This study provides evidence that coptisine, an ingredient of Unsei-in, prevents VSMC proliferation selectively at lower concentrations compared with various cells or other structurally related alkaloids.

CYTOTOXITY OF THE trans10,cis12 ISOMER OF CONJUGATED LINOLEIC ACID ON RAT HEPATOMA AND ITS MODULATION BY OTHER FATTY ACIDS, TOCOPHEROL, AND TOCOTRIENOL

This study was performed to evaluate the isomer-specific cytotoxic effects of conjugated linoleic acid (CLA) on rat hepatoma dRLh-84 cells in vitro. A 10trans,12cis (10t,12c)-CLA showed a strong cytotoxic effect on dRLh-84 cells in culture, whereas no such effect was observed with 9cis,11trans (9c,11t)-CLA or linoleic acid. The optimum concentration for induction of cytotoxicity by 10t,12c-CLA was 5 to 10 microM, but the effect was alleviated at higher concentrations. Coincubation with oleic or palmitoleic acid and 10t,12c-CLA cancelled the cytotoxic effect, but other major saturated or polyunsaturated fatty acids and eraidic acid did not interfere with 10t,12c-CLA-induced cytotoxity. The cytotoxic effect was also alleviated by alpha-tocopherol (alpha-toc) and alpha-tocotrienol but not by any other antioxidant reagent examined. Significant cytotoxicity of 10t,12c-CLA was detected after only a 15-min incubation, and the most noticeable effect was seen after 3 h. After incubation with 10t,12c-CLA at 10 microM, an additional 90 microM of 10t,12c-CLA or 100 microM of alpha-toc was also able to alleviate the cytotoxicity. When cells were treated with 10 microM 10t,12c-CLA for more than 48 h, treatment with additional CLA or alpha-toc could not prevent cell death.

Alleviation of the cytotoxic activity induced by trans10, cis12-conjugated linoleic acid in rat hepatoma dRLh-84 cells by oleic or palmitoleic acid

We showed the inhibitory effect of oleic and palmitoleic acids (OA and POA) on this trans10, cis12 (10 t, 12 c)-CLA induced cytotoxic activity. When cells were cultured in the presence of 10 t, 12 c-CLA, this potent cytotoxic effect on dRLh-84 cells was clearly obvious when compared to the control vehicle group. It was revealed that the levels of cellular OA and POA levels decreased upon cis9, trans11 (9 c,11 t) or 10 t, 12 c-CLA treatment in a time course dependent manner. OA or POA demonstrates a dose dependent inhibition of the 10 t, 12 c-CLA induced cytotoxicity. Notable nuclear fragmentation or activation of caspase-3 and 9 by 10 t, 12 c-CLA in dRLh-84 was counteracted by treatment with OA or POA. Results also suggest that 10 t, 12 c-CLA induced apoptosis can be inhibited by treatment with OA or POA.

Potent cytotoxic effect of the trans10, cis12 isomer of conjugated linoleic acid on rat hepatoma dRLh-84 cells

We evaluated the cytotoxic effect of conjugated linoleic acid (CLA) on rat hepatoma dRLh-84 cells in vitro. When cells were cultured in the presence of CLA, strong cytotoxic effect on dRLh-84 cells was recognized at 1 μM level compared to the control vehicle group, and trans10, cis12-CLA but not cis9, trans11-CLA was shown to be an active isomer for inducing this effect. Increase of the sub-G1 population and activation of caspase-3 and 9 accompanied with a time-dependent cleavage of poly(ADP–ribose) polymerase were recognized in dRLh-84 cells treated with trans10, cis12-CLA. In addition, we could see nuclear fragmentation in dRLh-84 cells treated with trans10, cis12-CLA by laser scanning confocal microscopy observation. Cytotoxic effect of trans10, cis12-CLA on normal hepatocytes was weaker than on dRLh-84 cells. These data indicate trans10, cis12-CLA has a potent cytotoxic effect on dRLh-84 cells through at least in part by an apoptotic pathway.

Dose-Dependent Effect of Dietary Conjugated Linoleic Acid on the Growth of Rat Hepatoma dRLh-84 Cells In Vivo

In this study, the effect of varying doses of conjugated linoleic acid (CLA) on the growth of transplanted hepatoma dRLh-84 cells and the relationship between tumor growth and prostaglandin (PG) E2 production or cyclooxygenase (COX)-2 expression were examined. Donryu rats were fed an experimental diet containing 0, 0.1, 0.5, or 2 wt.% CLA for 3 wk, and then dRLh-84 cells were transplanted into the liver. Results show that dietary CLA (0.5 and 2 wt.%) significantly enhanced the growth of the transplanted hepatoma cells compared to the non-CLA diet group at 20 d after cell transplantation. Tumor weight at 10 d after transplantation was also significantly higher in the 2 wt.% CLA group than in non-CLA fed rats. Ten days after transplantation, the PGE2 level in the tumor tissue was shown to be depressed in a CLA dose-dependent manner. Cyclooxygenase-2 (COX-2) mRNA expression in the tumor also tended to be lower in the CLA group than in the non-CLA diet group 10 d after transplantation. Dietary CLA did not affect the tumor phospholipid arachidonic acid level, which is a substrate for PG synthesis. These results indicate that dietary CLA of at least 0.5 wt.% enhances the growth of transplanted dRLh-84 cells in vivo. It is believed that growth promotion of dRLh-84 cells in vivo by CLA cannot be clarified by the PG synthesis dependent mechanism.

Effect of Dietary Conjugated Linoleic Acid on the In Vivo Growth of Rat Hepatoma dRLh-84

We examined the effect of dietary conjugated linoleic acid (CLA) on the growth of injected hepatoma dRLh-84 in Donryu rats. After experimental diets containing 0% or 2% CLA were given to male Donryu rats for 3 wk, dRLh-84 cells were injected into the left lobe of the hepatic capsule, and the experimental diet was continued. The cells formed a solid tumor ≥1 wk after the injection, and thereafter the tumor grew with feeding duration. In a morphological study, this tumor appeared to be a low-differentiated hepatoma, and there was no remarkable difference in the morphology of the tumor between 0% and 2% CLA groups. Tumor weight was significantly higher in the 2% CLA group than in the 0% CLA group throughout the feeding period after the injection. Serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities were significantly higher in 2% CLA-injected rats than in 0% CLA-injected rats at 3 wk after the injection. CLA upregulated acyl-CoA oxidase activity, especially 1 wk after the injection. However, dietary CLA did not activate carnitine palmitoyl transferase II, which is a rate-limiting enzyme in the mitochondrial β-oxidation pathway. Natural killer cell activity in the spleen tended to be higher in injected rats, but a significant effect of dietary CLA was not recognized. Serum interferon-γ and tumor necrosis factor-a levels were higher in injected than in sham rats. Moreover, these levels were higher in 2% CLA groups than in the respective 0% CLA groups.

Catalase contents in cells determine sensitivity to the apoptosis inducer gallic acid.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is known to induce apoptosis in cancer cells at lower IC50 values compared with values for normal cells. Apoptosis is inhibited completely by the addition of conditioned medium from cultured hepatocytes, whereas it is not prevented by conditioned media from tumor cells. We therefore studied the reason for the different response to GA-induced apoposis. GA-induced dRLh-84 cell death was completely abolished by the addition of peroxisome or cytosol as well as conditioned medium from primary cultured rat hepatocyte. As GA-induced cell death is known to be mediated by reactive oxygen species (ROS) and intracellular Ca2+, we determined the type of ROS generated by GA and found that GA generated hydrogen peroxide in culture medium. The addition of hydrogen peroxide generated by GA induced cell death in dRLh-84 cells. These results suggest that GA-induced cell death is mediated by hydrogen peroxide. On the other hand, the inhibitory activity of hepatocyte medium on GA-induced cell death was completely abolished by anti-catalase antibody. When the amount of catalase antigen was determined by Western blotting analysis, conditioned medium and the cytoplasm of hepatocytes contained high concentrations of catalase. Conditioned media from various tumor cell lines did not contain catalase, and the cytoplasm contained only low levels of catalase. These results show that GA-sensitive cells, including various tumor cells, produce only small amounts of catalase and secreted little enzyme into media, suggesting a lack of protective machinery against GA. In contrast, GA-insensitive cells, including hepatocytes, produce large amounts of catalase and release it in medium, resulting in the development of insensitivity to GA. In conclusion, catalase contents in cells determine different sensitivity to GA.

Mechanism of liver-selective thyromimetic activity of SK&F L-94901: evidence for the presence of a cell-type-specific nuclear iodothyronine transport process.

The thyromimetic compound SK&F L-94901 shows more potent thyromimetic activity in the liver than in the pituitary gland or heart when administered to rats. The mechanisms of liver-selectivity of SK&F L-94901 were examined using cultured rat hepatoma cells (dRLH-84) and rat pituitary tumor cells (GH3), both of which showed saturable cellular uptake of tri-iodothyronine (T(3)). When isolated nuclei with partial disruption of the outer nuclear membrane were used, SK&F L-94901 competed for [(125)I]T(3) binding to nuclear receptors almost equally in dRLH-84 and GH3 cells. SK&F L-94901 also did not discriminate thyroid hormone receptors (TR) alpha1 and beta1 in terms of binding affinity and activation of the thyroid hormone responsive element. In intact cells, however, SK&F L-94901 was a more potent inhibitor of nuclear [(125)I]T(3) binding in dRLH-84 cells than in GH3 cells at an early phase of the nuclear uptake process and after binding equilibrium. These data suggest that SK&F L-94901 is more effectively transported to nuclear TRs in hepatic cells than in pituitary cells and therefore shows liver-selective thyromimetic activity. In conclusion, SK&F L-94901 discriminates hepatic cells and pituitary cells at the nuclear transport process. The cellular transporters responsible for this discrimination were not evident.

Increased expression of Schwannoma-derived growth factor (SDGF) mRNA in rat tumor cells: Involvement of SDGF in the growth promotion of rat gliomas

Schwannoma-derived growth factor (SDGF) is a member of the epidermal growth factor (EGF) family, having mitogenic activity on rat astrocytes, fibroblasts and Schwann cells. The SDGF gene is significantly expressed in the newborn rat lung and in the adult rat sciatic nerve. However, except for one rat schwannoma cell line, from which SDGF and its cDNA were isolated, nothing is known about SDGF expression in established tumor cell lines. We examined the expression level of the SDGF gene in a variety of rat tumor cell lines by Northern blotting and found that it was increased in 11 of 25 established lines. The most abundant SDGF mRNA, which was about 50-fold higher than in the newborn rat lung, was expressed in rat liver adenoma dRLa74 cells. In rat glioma cell lines, such as C6, 9L and T9, and in the rat hepatoma dRLh84 and H411E cells, the SDGF expression level was about 10-fold higher than in the newborn rat lung. In 8 of 13 cell lines expressing SDGF mRNA, the EGF receptor (EGFR) gene, the product of which is regarded as a functional receptor of SDGF, was co-expressed. In addition, transfected gene-dependent anti-sense SDGF RNA expression under the control of the human metallothionein promoter significantly suppressed the in vitro growth as well as in vivo tumorigenicity of 9L glioma cells. Our results suggest that SDGF acts as an autocrine growth factor in the development and growth of rat tumors such as gliomas.

Alternative splicing of the pyruvate kinase M gene in a minigene system.

The M1-type and M2-type isozymes of pyruvate kinase are produced from a single gene by mutually exclusive use of exons 9 and 10. Selection of exon 10 generates the M2 type, which occurs in most tissues, whereas the M1 type is expressed by use of exon 9 only in skeletal muscle, heart and brain. We investigated the mechanism by which exon 10, but not exon 9 is selected in M2-expressing cells by transfecting minigenes containing exon 9 and/or exon 10 in cells and by analyzing the transcripts using reverse-transcriptase polymerase chain reaction. Deletion of the most conserved region in intron 8 did not affect selection of exon 10 in dRLh-84 cells, which express only the M2 type. Exclusion of exon 10 from the minigene resulted in two major spliced products. One included correctly spliced exon 9 and the other skipped this exon. Similar splicing patterns were observed when these minigenes were transfected in hepatocytes which express the L type, but not M1 or M2 types. The 5' splice site but not the 3' splice site of exon 9 was found to be hardly recognized by the splicing machinery in dRLh-84 cells. Mutation of the 5' splice site sequence of exon 9 to that of exon 10 and vice versa did not change the splicing patterns. However, mutation of this site of exon 9 to a perfectly complementary sequence of U1 snRNA resulted in selection of exon 9 correctly spliced to exon 10. A 9-10 fusion exon (constructed by substitution of 68 bases of the 3' portion of exon 9 and 33 bases of the 5' portion of intron 9 for the corresponding regions of exon 10 and intron 10) was also correctly incorporated into a major product together with exon 10. Thus, we propose that exon 9 is not recognized in non M1-expressing cells due to the weak signal of its 5' splice site and that, although the 5' splicing signal of exon 10 also appears to be weak, this exon can be recognized in these cells because the 5' recognition signal may be relatively strengthened by cis-acting element(s) which may be present in the 3' portion of exon 9 and the 5' portion of intron 9 and/or the corresponding regions of exon 10 and intron 10.

Selective induction of cell death in cancer cells by gallic acid.

Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally occurring plant phenol obtained by the hydrolysis of tannins and is know to show some pharmacological activities. In screening anti-cancer agents in traditional Chinese medicines, gallic acid was found to show cytotoxicity against all cancer cells that we examined in this study (IC50s: 4.8-13.2 micrograms/ml). Gallic acid was found to show cytotoxicity against primary cultured rat hepatocytes and macrophages, and lesser cytotoxicity against fibroblasts and endothelial cells. Cell death in dRLh-84 cells occurred within 6h after gallic acid treatment at a concentration of more than 20 micrograms/ml. A study of structurally related compounds suggested that the cytotoxicity shown by gallic acid was not a common feature in phenolic compounds, but was a fairly specific characteristic of gallic acid. That is, three adjacent phenolic hydroxyl groups of gallic acid were responsible for the cytotoxicity, and the carboxyl group was not responsible, but seemed to be implicated in distinguishing between normal cells and cancer cells.