Novikoff Hepatoma Research Articles

RBM45 is an m6A-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

SUMMARYN6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are incompletely understood. RBM45 acts as an m6A-binding protein that is highly expressed during neurodevelopment. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings show a role for RBM45 in controlling mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

Preparation of gelatin/Ag NPs under ultrasound condition: A potent and green bio-nanocomposite for the treatment of pleomorphic hepatocellular carcinoma, morris hepatoma, and novikoff hepatoma

Regarding applicative, facile, green chemical research, a bio-inspired approach is being reported for the sonochemical synthesis of Ag nanoparticles by gelatin as a natural reducing/stabilizing and solid support agent without using any toxic and harmful reagent. The biosynthesized Ag NPs@gelatin bionanocomposite were characterized by advanced physicochemical techniques like ultraviolet–visible (UV–Vis), Fourier Transformed Infrared spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), High-Resolution Transmission Electron Microscopy (HR-TEM), Energy Dispersive X-ray spectroscopy (EDX), and X-ray Diffraction (XRD) study. It has been established that gelatin-stabilized silver nanoparticles have a spherical shape with a mean diameter from ∼5–10 nm. To survey the antioxidant potentials of Ag NPs@gelatin, one of the common antioxidant techniques i.e., DPPH was used. The results of DPPH test proved excellent antioxidant properties of Ag NPs@gelatin in comparison to the positive control used i.e., butylated hydroxytoluene. The IC50 of Ag NPs@gelatin was 250 µg/mL in the antioxidant test. Determination of anti-liver cancer effects of Ag NPs@gelatin was carried out by the MTT assay and against pleomorphic hepatocellular carcinoma (SNU-387), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) cell lines. The results of MTT assay confirmed removing SNU-387, McA-RH7777, and N1-S1 Fudr cell lines after treating with low concentrations of Ag NPs@gelatin. The IC50 of the Ag NPs@gelatin was 192, 144, and 228 µg/mL against SNU-387, McA-RH7777, and N1-S1 Fudr cell lines, respectively. The best result of anti-human liver cancer effects of Ag NPs@gelatin against the above cell lines was seen in the case of the N1-S1 Fudr cell line. According to the above results, significant anti-liver cancer effects of Ag NPs@gelatin is confirmed.

One-pot preparation of nano-scaled magnetic-pectin particles (Fe3O4@pectin NPs): cytotoxicity, antioxidant, and anti-liver cancer properties

This work described the one-pot synthesis of orange pectin encapsulated Fe3O4 nanoparticles (Fe3O4@Pectin NPs) which is prepared by co-precipitation of Fe(II/(III) ions in alkaline solution mediated by pectin. This process led to formation of magnetic nanoparticles within the network of pectin. Physicochemical characterization of the as-synthesized Fe3O4@Pectin NPs was carried out through Fourier transformed infrared spectroscopy (FT-IR), electron microscopy (SEM and TEM), energy dispersive X-ray spectroscopy (EDX), vibrating sample magnetometer (VSM) and X-ray diffraction (XRD). The in vitro cytotoxic and anti-liver cancer effects of biologically synthesized Fe3O4@Pectin NPs against pleomorphic hepatocellular carcinoma (SNU-387), hepatic ductal carcinoma (LMH/2A), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) cancer cell lines were assessed. The anti-liver cancer properties of the Fe3O4@Pectin NPs could significantly remove pleomorphic hepatocellular carcinoma (SNU-387), hepatic ductal carcinoma (LMH/2A), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) cancer cell lines in a time and concentration-dependent manner by MTT assay. The IC50 of the Fe3O4@Pectin NPs were 8, 13, 10, and 7 µg/mL against pleomorphic hepatocellular carcinoma (SNU-387), hepatic ductal carcinoma (LMH/2A), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) cancer cell lines. The antioxidant activity of Fe3O4@Pectin NPs was determined by DPPH method. The Fe3O4@Pectin NPs showed the high antioxidant activity according to the IC50 value. It seems that the anti-human liver cancer effect of recent nanoparticles is due to their antioxidant effects.

Green formulation of Ag nanoparticles by Hibiscus rosa-sinensis: Introducing a navel chemotherapeutic drug for the treatment of liver cancer

An eco-friendly biosynthesized Ag NPs immobilized Hibiscus rosa-sinensis extract has been introduced. The as-prepared nanoparticles were characterized using UV–Vis, SEM, and FT-IR analysis. In the FT-IR test, the presence of many antioxidant compounds with related bonds caused the excellent condition for reducing of silver in the silver nanoparticles. In UV–Vis, the clear peak in the wavelength of 428 nm indicated the formation of silver nanoparticles. The synthesized nanoparticles had very low cell viability and high anti-liver cancer activities dose-dependently against pleomorphic hepatocellular carcinoma (SNU-387), hepatic ductal carcinoma (LMH/2A), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) cell lines without any cytotoxicity on the normal cell line (HUVEC). The synthesized nanoparticles inhibited half of the DPPH molecules in the concentration of 78 µg/mL. Perhaps notable anti-liver cancer activities of the synthesized nanoparticles against common liver cancer cell lines are linked to their antioxidant activities.

Iron nanoparticles green-formulated by Coriandrum sativum leaf aqueous ‎extract: Investigation of its anti-liver cancer effects

IntroductionRecently, scientists have ‎understood that metallic nanoparticles especially iron nanoparticles have ‎excellent anticancer properties‎. A green, productive, and environmentally method was developed for the valuable study and the effective preparation of the biosynthesis of iron nanoparticles using aqueous extracts from the leaf of Coriandrum sativum as a result of reducing and stabilizing factor. The simplicity of the reaction, heterogeneous system, and easy work up are the benefits of the present method.Material and methodsThe as-prepared nanoparticles (FeNPs) was characterized using UV-Vis, SEM, and FT-IR. It has ‎been shown that the iron nanoparticles have spherical shape and uniform size.ResultsThe synthesized nanoparticles had very low cell viability and high anti-liver cancer activities ‎dose-dependently against pleomorphic hepatocellular carcinoma (SNU-387), hepatic ductal ‎carcinoma (LMH/2A), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) ‎cell lines without any cytotoxicity on the normal cell line (HUVEC). The synthesized ‎nanoparticles inhibited half of the DPPH molecules in the concentration of 132 µg/mL. Perhaps ‎notable anti-liver cancer activities of the synthesized nanoparticles against common liver cancer ‎cell lines are linked to their antioxidant activities. ‎ConclusionsOur results point out that the FeNPs from Coriandrum sativum extract are apposite stabilizing ‎agents, which serve as an effective anticancer agent against liver cancer cell lines.‎

The effect of nutmeg essential oil constituents on Novikoff hepatoma cell viability and communication through Cx43 gap junctions

Essential oils from plants are a potential source of molecules having anti-inflammatory, anticancer, cardiotropic, and other activities. However, most of these effects lack mechanistic explanations and structure–activity relationship testing. In the present study, we: 1) identified the nutmeg essential oil (NEO) composition; 2) using molecular docking, we determined the putative regulatory binding sites on the connexin 43 (Cx43) that is responsible for gap junction-dependent intercellular communication (GJIC) in the majority of tissues; 3) examined the effect of NEO and its three constituents – sabinene, α-pinene, and α-copaene – on GJ conductance and gating in Novikoff cells expressing endogenous Cx43; and 4) verified whether NEO effects on GJIC correlated with its action on Novikoff cell viability, proliferation, and colony formation capability. Our results revealed NEO and its constituents as potent and efficient Cx43 GJ inhibitors acting by slow gating mechanism. In addition, NEO reduced Novikoff hepatoma cell viability, proliferation, and colony formation capability; however, this was achieved at higher doses and was unrelated to its effects on GJIC.

Celecoxib enhances the therapeutic efficacy of epirubicin for Novikoff hepatoma in rats.

Epirubicin is a chemotherapy agent for hepatocellular carcinoma (HCC). However, the outcome of HCC patients receiving epirubicin remains unsatisfactory. Moreover, our previous study indicated that celecoxib suppresses HCC progression and liver cancer stemness. This study evaluated the potential of celecoxib to serve as a complementary therapy during epirubicin treatment. Cell proliferation, apoptosis, invasiveness, and anchorage‐independent growth were analyzed in hepatoma cells. Therapeutic efficacy was validated in rat orthotopic Novikoff hepatoma. After animal sacrifice, the antitumor mechanism of celecoxib and epirubicin combined therapy was investigated by histological analysis. Celecoxib enhanced the cytotoxic activity of epirubicin in HCC cells by promoting apoptosis. Besides, celecoxib potentiated the antineoplastic function of epirubicin in inhibiting the invasiveness and anchorage‐independent growth of HCC cells. Ultrasound monitoring showed that combined therapy was more potent than either therapy alone in perturbing HCC progression. Consistently, the size and weight of dissected HCC tissues from rats receiving combined therapy were smallest among all groups. HCC treated with combined therapy exhibited the highest prevalence of apoptotic cells, which was accompanied by reduced proliferating and angiogenic activities in tumor tissues. Moreover, the expression levels of cancer stemness markers (CD44 and CD133) and drug transporter MDR‐1 were significantly diminished in rats receiving combined therapy. Besides, celecoxib treatment increased the infiltration of cytotoxic T lymphocytes (CTLs) and reduced the number of regulatory T cells (Tregs), tumor‐associated macrophages (TAMs), and the expression of immune checkpoint PD‐L1 in HCC tissues during epirubicin therapy. Celecoxib augmented the therapeutic efficacy while modulated cancer stemness and antitumor immunity. Thus, celecoxib may serve as complementary therapy to improve the outcome of patients with advanced HCC during epirubicin treatment.

Celecoxib suppresses hepatoma stemness and progression by up-regulating PTEN.

Celecoxib, a COX-2 inhibitor and non-steroidal anti-inflammatory drug, can prevent several types of cancer, including hepatocellular carcinoma (HCC). Here we show that celecoxib suppressed the self-renewal and drug-pumping functions in HCC cells. Besides, celecoxib depleted CD44+/CD133+ hepatic cancer stem cells (hCSC). Prostaglandin E2 (PGE2) and CD133 overexpression did not reverse the celecoxib-induced depletion of hCSC. Also, celecoxib inhibited progression of rat Novikoff hepatoma. Moreover, a 60-day celecoxib program increased the survival rate of rats with hepatoma. Histological analysis revealed that celecoxib therapy reduced the abundance of CD44+/CD133+ hCSCs in hepatoma tissues. Besides, the hCSCs depletion was associated with elevated apoptosis and blunted proliferation and angiogenesis in hepatoma. Celecoxib therapy activated peroxisome proliferator-activated receptor γ (PPARγ) and up-regulated PTEN, thereby inhibiting Akt and disrupting hCSC expansion. PTEN gene delivery by adenovirus reduced CD44/CD133 expression in vitro and hepatoma formation in vivo. This study suggests that celecoxib suppresses cancer stemness and progression of HCC via activation of PPARγ/PTEN signaling.

Gas challenge–blood oxygen level-dependent (GC-BOLD) MRI in the rat Novikoff hepatoma model

Purpose The purpose of the study was to investigate the relationship between gas challenge–blood oxygen level-dependent (GC-BOLD) response angiogenesis and tumor size in rat Novikoff hepatoma model. Materials and Methods Twenty adult male Sprague–Dawley rats (weighting 301–325 g) were used for our Animal Care and Use Committee-approved experiments. N1-S1 Novikoff hepatomas were grown in 14 rats with sizes ranging from 0.42 to 2.81 cm. All experiments were performed at 3.0 T using a custom-built rodent receiver coil. A multiple gradient-echo sequence was used for R2 ⁎ measurements, first during room air (78% N 2/20% O 2) breathing and then after 10 min of carbogen (95% O 2/5% CO 2) breathing. After image acquisition, rats were euthanized, and the tumors were harvested for histological evaluation. Results The R2 ⁎ change between air and carbogen breathing for small hepatomas was positive; R2 ⁎ changes changed to negative values for larger hepatomas. We found a significant positive correlation between tumor R2 ⁎ change and tumor microvessel density (MVD) ( r=0.798, P=.001) and a significant inverse correlation between tumor R2 ⁎ change and tumor size ( r=−0.840, P<.0001). Conclusions GC-BOLD magnetic resonance imaging measurements are well correlated to MVD levels and tumor size in the N1-S1 Novikoff hepatoma model; GC-BOLD measurements may serve as noninvasive biomarkers for evaluating angiogenesis and disease progression and/or therapy response.

Rapid induction of orthotopic hepatocellular carcinoma in immune-competent rats by non-invasive ultrasound-guided cells implantation

BackgroundThe fact that prognoses remain poor in patients with advanced hepatocellular carcinoma highlights the demand for suitable animal models to facilitate the development of anti-cancer medications. This study employed a relatively non-invasive approach to establish an orthotopic hepatocellular carcinoma model in immune-competent rats. This was done by ultrasound-guided implantation of cancer cells and the model was used to evaluate the therapeutic efficacy of short-term and low-dose epirubicin chemotherapy.MethodsRat Novikoff hepatoma cells were injected percutaneously into the liver lobes of Sprague-Dawley rats under the guidance of high resolution ultrasound. The implantation rate and the correlation between dissected and ultrasound-measured tumor sizes were evaluated. A similar induction procedure was performed by means of laparotomy in a different group of rats. Pairs of tumor measurement were compared by ultrasound and computerized tomography scan. Rats with a successful establishment of the tumor were divided into the treatment (7-day low-dose epirubicin) group and the control group. The tumor sizes were non-invasively monitored by the same ultrasound machine. Blood and tumor tissues from tumor-bearing rats were examined by biochemical and histological analysis respectively.ResultsUltrasound-guided implantation of Novikoff hepatoma cells led to the formation of orthotopic hepatocellular carcinoma in 60.4% (55/91) of the Sprague-Dawley rats. Moreover, tumor sizes measured by ultrasound significantly correlated with those measured by calipers after sacrificing the animals (P < 0.00001). The rate of tumor induction by ultrasound-guided implantation was comparable to that of laparotomy (55/91, 60.4% vs. 39/52, 75%) and no significant difference in sizes of tumor was noted between the two groups. There was a significant correlation in tumor size measurement by ultrasound and computerized tomography scan. In tumor-bearing rats, short-term and low-dose epirubicin chemotherapy caused a significant reduction in tumor growth, and was found to be associated with enhanced apoptosis and attenuated proliferation as well as a decrease in the microvessel density in tumors.ConclusionsUltrasound-guided implantation of Novikoff hepatoma cells is an effective means of establishing orthotopic hepatocellular carcinoma in Sprague-Dawley rats. Short-term and low-dose epirubicin chemotherapy had perturbed tumor progression by inducing apoptosis and neovascularization blockade.

Superiority of combined chemo-embolization and portal infusion with 5-fluorouracil over locoregional infusion concepts in Novikoff hepatoma-bearing rats

The aim of this study was to investigate experimentally whether there is a superior effect of the combination of hepatic artery chemo-embolization with portal vein infusion over either of the two treatment modalities alone. Novikoff hepatoma cells transplanted under the liver capsule of Sprague Dawley rats were used as a model. Tumor growth was assessed at 7 and 21 days after tumor inoculation. The prolamine solution Ethibloc was employed for embolization, and 5-fluorouracil was used as a chemotherapeutic agent for both infusion and chemo-embolization. All arterial treatment modalities were administered in a super-selective manner. There was no intolerable toxicity after dosages of 55 to 125 mg 5-fluorouracil/kg body weight. With regard to therapeutic efficacy the results show that embolization is an effective therapeutic means for inducing tumor necrosis in selected liver areas. As a consequence, the ranking of all treatment modalities was based on the combined evaluation of tumor size and extent of tumor necrosis. According to this evaluation, hepatic artery chemo-embolization was superior to the respective type of infusion (P<0.01). In addition, the combination of both modalities in the form of hepatic artery chemo-embolization and portal vein infusion was effective in destroying more than 97% of vital tumor tissue (P<0.01). These results suggest the need for a comparative clinical study.

Gap Junctions Assemble in the Presence of Cytoskeletal Inhibitors, but Enhanced Assembly Requires Microtubules

The role of cytoskeletal elements in gap junction (GJ) assembly has been studied using Novikoff hepatoma cells treated with cytochalasin B (CB) to disrupt actin filaments or with colchicine or nocodazole to disrupt microtubules. After 60 min of cell reaggregation, freeze-fracture was used to evaluate quantitatively the “initiation,” “maturation,” and “growth” phases of GJ assembly. The development of junctional permeability to fluorescent dyes was also analyzed. The only effects of CB on the structure or permeability of the developing junctions involved an elongation of GJ aggregates and a small decrease in formation plaque areas. Colchicine (but not the inactive form, lumicolchicine) prevented the enhancement of GJ growth by cholesterol, but its effect on basal growth was equivocal. Nocodazole inhibited the growth of GJ, even under basal conditions, without an effect on initiation. Nocodazole also blocked the forskolin-enhanced increase in the growth of GJs and, in living MDCK cells, reduced the movement of transport intermediates containing green fluorescent protein-tagged connexin43. Thus, neither actin filaments nor microtubules appear to restrict GJ assembly by anchoring intramembrane GJ proteins, nor are they absolutely required for functional GJs to form. However, microtubules are necessary for enhanced GJ growth and likely for facilitating connexin trafficking under basal conditions.

Gap junction assembly: PTX-sensitive G proteins regulate the distribution of connexin43 within cells.

Cells expressing connexin43 are able to upregulate gap junction (GJ) communication by enhancing the assembly of new GJs, apparently through increased connexin trafficking. Because G proteins are known to regulate different aspects of protein trafficking, we examined the effects of pertussis toxin (PTX; a specific inhibitor of certain G proteins) on GJ assembly. Dissociated Novikoff hepatoma cells were reaggregated for 60 min to form nascent junctions. PTX inhibited GJ assembly, as indicated by a reduction in dye transfer. Electron microscopy also revealed a 60% decrease in the number of GJ channels per cell interface. Importantly, PTX blocked the twofold enhancement in GJ assembly found in the presence of low-density lipoprotein. Two G(i alpha) proteins (G(i alpha 2) and G(i alpha 3)), which have been implicated in the control of membrane trafficking, reacted with PTX in ADP-ribosylation studies. PTX and/or the trafficking inhibitors, brefeldin A and monensin, inhibited GJ assembly to comparable degrees. In addition, assays for GJ hemichannels demonstrated reduced plasma membrane levels of connexin43 following PTX treatment. These results suggest that PTX-sensitive G proteins regulate connexin43 trafficking, and, as a result of inhibition with PTX, the number of plasma membrane hemichannels available for GJ assembly is reduced.

A mutation in subunit B of the DNA polymerase alpha-primase complex from Novikoff hepatoma cells concomitant with a conformational change and abnormal catalytic properties of the DNA polymerase alpha-primase complex.

Mutated constituents of the DNA replication complex might contribute to the mutational load of the genome during tumor development by impairing DNA synthesis as well as cell cycle-related control of DNA replication. To prove or disprove this hypothesis, we looked for mutations in the cDNA sequences of the four subunits of DNA polymerase alpha-primase from both highly malignant Novikoff hepatoma cells and regenerating normal rat liver and compared physicochemical and catalytic properties of the DNA polymerase alpha-primase complexes purified from both sources. Sequence analysis showed two mutations in subunit B from Novikoff cells: one in nucleotide position 855 (CCG-->CCA) that did not result in an amino acid exchange and one in position 862 (GTG-->ATG) that caused a change of valine to methionine in codon 288. No mutation was found in the three other subunits. The wild-type and mutated sequences of subunit B were cloned and expressed in vitro. Sedimentation analysis of the expressed polypeptides revealed different sedimentation constants, indicating that the amino acid exchange affected the conformation of subunit B. The analysis of the purified DNA polymerase alpha-primase complexes showed a sedimentation value that was significantly higher for the enzyme complex from normal liver than for that from Novikoff cells. In addition, DNA polymerase alpha-primase complexes from Novikoff cells showed higher sensitivity to camptothecin, topotecan, and structurally related compounds (such as (R,S)-7-ethyl-10-hydroxy camptothecin, 9-aminocamptothecin, and 10-hydroxycamptothecin) than the enzyme from normal rat liver. Thus, the amino acid change found in subunit B appears to result in a conformational change of the DNA polymerase alpha-primase complex from Novikoff hepatoma cells. Whether this mutation influences genetic instability or tumor development needs to be explored.

Microscopic Analysis of Oxidative Stress in Cultured Cells. II. Transmission Electron Microscopy

Abstract The insulin-sensitizing agent troglitazone (Rezulin) has been shown to cause severe hepatotoxicity and liver failure. While the toxic mechanism is still unknown, a component may be oxidative stress. We have demonstrated that increased lipid peroxidation, decreased glutathione levels, and collapse in mitochondrial membrane potential occurred following exposure of cultured hepatocytes to troglitazone. These hallmarks of oxidative stress can manifest morphologically in several ways including damage to cellular membranes, mitochondrial abnormalities, and cell death. The purpose of this study was to assess morphologic changes caused by exposure to troglitazone or cumene hydroperoxide (CH), a compound known to induce oxidative stress. HepG2 and Novikoff hepatoma cells were grown on poly-L-lysine coated 6-well plates overnight, and exposed to 10, 25, 50 or 100 μM concentrations of troglitazone or CH in the growth media for 2 hours. Cells were fixed with 2.5% glutaraldehyde in phosphate buffer, harvested, and pelleted. Pellets were post-fixed in 1% osmium tetroxide, and processed to epoxy resin.

Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking.

Given the rapid turnover of connexin proteins, gap junction (GJ) assembly represents an important means of regulating the extent of GJ communication between cells. This report describes an increase in the level of GJ assembly within one hour following treatment with cAMP-elevating reagents or low density lipoprotein (LDL). Dye transfer methods and freeze-fracture with electron microscopy were used to assay junctional permeability and structure, respectively, subsequent to the dissociation, recovery and reaggregation of Novikoff hepatoma cells. Reaggregating cells in the presence of agents that increase cAMP levels (8-Br-cAMP, forskolin and IBMX) enhanced both dye transfer rates between cells and the extent of GJ formation 2- to 3-fold. These data and studies with the protein kinase A inhibitor, H-89, indicate that cAMP signaling plays a key role in enhanced assembly. The response to LDL parallels that to cAMP and relies on the activity of both adenylyl cyclase and protein kinase A. Immunoblot analysis revealed no change in the level of connexin43 (Cx43) or its phosphorylation states over a period of 2.5 hours. However, three agents (brefeldin A, monensin and nocodazole), that inhibit intracellular membrane trafficking by different mechanisms, all blocked the enhanced assembly of GJs when triggered by either elevated cAMP or exposure to LDL. Related studies, which employed trafficking inhibitors at different stages in GJ assembly, suggested that Cx43 trafficking during enhanced assembly is regulated, in part, by cell contact. Intracellular sources of Cx43 were characterized by colabeling for several markers of cytoplasmic membrane systems. We conclude that an increase in GJ assembly: (i) occurs rapidly in the presence of elevated cAMP or LDL, (ii) does not require an increase in Cx43 levels or major changes in Cx43 phosphorylation and (iii) is dependent upon the trafficking of Cx43 from intracellular storage sites.

The efficacy of adjuvant cytostatic therapy after organ transplantation for malignancy: an experimental study with a combined transplantation/tumor model.

New data show that perioperative cytostatic therapy is beneficial in the case of liver transplantation for hepatic cancer. However, it has not been established clearly whether chemotherapy interferes with graft rejection. We therefore studied the interactions between tumor growth and graft rejection, especially with regard to chemotherapy, using a combined tumor/transplantation model. As a tumor model, we used the Novikoff hepatoma, a malignant hepatoma that was injected subcutaneously into the backs of rats. Heterotopic heart grafting served as the transplantation model. In a first step (a), we studied the effect of cytostatic therapy on tumor growth: tumor cells were injected, and in four groups epirubicin, cyclosporine, epirubicin + cyclosporine, and placebo were applied, in corresponding groups, transplantation was additionally performed. Tumor growth was measured and the resected tumors were examined by histology and immunohistology. In a second step (b), we studied the effect of chemotherapy on graft rejection: transplantation was performed and the above-mentioned drugs were applied; in corresponding groups, a solid tumor was additionally induced and resected immediately before transplantation. The results of these procedures were as follows: (a) Epirubicin decreased tumor growth and diminished the volume-increasing effect of cyclosporine significantly. After transplantation, tumor growth was similar. (b) Epirubicin prolonged graft survival significantly, and the combination with cyclosporine had an augmenting effect. In the corresponding groups, graft survival was similar. In conclusions. chemotherapy diminishes the tumor-increasing effect of cyclosporine and does not interfere negatively with graft survival. It might therefore be beneficial after transplantation for malignancy.

The efficacy of adjuvant cytostatic therapy after organ transplantation for malignancy: an experimental study with a combined transplantation/tumor model

Abstract New data show that perioperative cytostatic therapy is beneficial in the case of liver transplantation for hepatic cancer. However, it has not been established clearly whether chemotherapy interferes with graft rejection. We therefore studied the interactions between tumor growth and graft rejection, especially with regard to chemotherapy, using a combined tumor/transplantation model. As a tumor model, we used the Novikoff hepatoma, a malignant hepatoma that was injected subcutaneously into the backs of rats. Heterotopic heart grafting served as the transplantation model. In a first step (a), we studied the effect of cytostatic therapy on tumor growth: tumor cells were injected, and in four groups epirubicin, cyclosporine, epirubicin + cyclosporine, and placebo were applied, in corresponding groups, transplantation was additionally performed. Tumor growth was measured and the resected tumors were examined by histology and immunohistology. In a second step (b), we studied the effect of chemotherapy on graft rejection: transplantation was performed and the above-mentioned drugs were applied; in corresponding groups, a solid tumor was additionally induced and resected immediately before transplantation. The results of these procedures were as follows: (a) Epirubicin decreased tumor growth and diminished the volume-increasing effect of cyclosporine significantly. After transplantation, tumor growth was similar. (b) Epirubicin prolonged graft survival significantly, and the combination with cyclosporine had an augmenting effect. In the corresponding groups, graft survival was similar. In conclusions. chemotherapy diminishes the tumor-increasing effect of cyclosporine and does not interfere negatively with graft survival. It might therefore be beneficial after transplantation for malignancy.

Enhancement of 5-Fluorouracil Anabolism by Methotrexate and Trimetrexate in Two Rat Solid Tumor Models, Walker 256 Carcinosarcoma and Novikoff Hepatoma, as Evaluated by 19F-Magnetic Resonance Spectroscopy

Although 5-fluorouracil (5-FU) is one of the most effective single agents in treating solid tumors, its low effectiveness as a single agent has led to development of a number of modulators intended to enhance its therapeutic effectiveness. Of these, methotrexate (MTX) and trimetrexate (TMTX) have been shown to have synergistic anticancer activity with 5-FU. The effect of these two drugs on the uptake and the intratumoral metabolism of 5-FU was studied in two rat tumor models using 19F-nuclear magnetic resonance spectroscopy: on excised samples of Walker 256 carcinosarcoma and noninvasively (in vivo) in Novikoff hepatoma. In the rats bearing the Walker 256 tumor, a 4-hr pretreatment with MTX showed the maximal increase in the rate of conversion from 5-FU to its fluorinated nucleotides/nucleosides. In the rats bearing the Novikoff hepatoma, both modulators increased the amounts of cyctotoxic anabolites of 5-FU, but at the doses administered, the cumulative amounts of 5-FU anabolites formed after MTX were significantly higher than those formed after TMTX or after saline control. On the other hand, the increase in the levels of the fluorinated nucleotides/nucleosides after TMTX peaked at a later time. The possible significance of these findings is that timing of administration of a modulator is important because it affects both transport and metabolism of 5-FU. The two modulators studied, both antifolates, act differently on transport and on metabolism: MTX affects both, whereas TMTX, at the level studied, appears to affect predominantly the metabolic process. In addition, significant differences exist between tumor models. These data suggest possible mechanisms and processes that should be studied further in humans, using these noninvasive pharmacokinetic imaging methods for monitoring 5-FU targeting and metabolism.

A mutation detected in DNA polymerase delta cDNA from Novikoff hepatoma cells correlates with abnormal catalytic properties of the enzyme.

Tumor development is characterized by accumulation of mutations. Such mutations, if induced by carcinogens in DNA polymerase genes, would confer mutator properties on the DNA replication machinery, even at later stages of development. To investigate whether DNA polymerase delta can be mutated, we compared these enzymes from highly malignant Novikoff hepatoma cells and from regenerating normal rat liver. We sequenced the DNA polymerase delta cDNA from both sources and investigated the physico-chemical properties, inhibition characteristics, and copying fidelity of the purified enzymes. The cDNA sequences examined included the entire reading frame encoding the catalytic subunit (subunit I) of DNA polymerase delta. First-strand cDNAs were prepared from total RNA of both normal rat liver and Novikoff cells by reverse transcription, and the polymerase delta sequences were amplified by the polymerase chain reaction. cDNA (3325 bp) were sequenced. A single heterozygous mutation (CGG --> CAG) has been detected in nucleotide position 1948 (codon 648) of the polymerase delta gene from Novikoff cells, resulting in an Arg to Gln change. Position 648 lies just proximal to the conserved region VI, which is part of the "fingers" subdomain of alpha-like polymerases. This subdomain is involved in dNTP binding. Upon comparison of biochemical characteristics of partially purified DNA polymerase delta from both Novikoff cells and rat liver, the following properties of the enzyme from Novikoff cells were found to be altered: (i) K(50) values for nucleotide analogs (e.g. butylphenyl-dGTP) were lower, (ii) sensitivity to various antineoplastic drugs (e.g. doxorubicin, topotecan and distamycin) was enhanced, (iii) copying fidelity was decreased when primer templates containing O(6)-methylguanine were used, and (iv) the activity of DNA polymerase delta from Novikoff tumor cells was less stimulated by lactate dehydrogenase than the enzyme from normal cells. The altered biochemical characteristics of DNA polymerase delta from Novikoff cells suggest mutator properties. We conclude that the point mutation detected in the cDNA might be causally related to the observed changes in inhibition characteristics and copying fidelity.