Yoshida AH-130 Ascites Hepatoma Research Articles

Cancer Cachexia, Malnutrition, and Tissue Protein Turnover in Experimental Animals

The role of malnutrition in the development of cachexia in rats bearing the Yoshida ascites hepatoma AH-130 was investigated by comparing the changes in tissue protein turnover in these animals with those observed in pair-fed controls. The tumor elicited in rats an early and conspicuous loss of body weight and tissue waste. Protein loss was particularly prominent for the gastrocnemius muscle and the heart and less pronounced for the soleus, while the diaphragm was little affected. Liver, kidneys, and spleen transiently increased in weight then regressed and eventually atrophied, while adrenals were enlarged over the whole experimental period. Protein waste was mainly due to acceleration of tissue protein breakdown, this protein hypercatabolic state being associated with increased cathepsin D activity in liver and gastrocnemius. In pair-fed animals the liver showed a marked protein loss resulting from enhanced catabolism, while the sharp decrease of heart protein content and the less prominent waste of the gastrocnemius were due to a reduction in protein synthesis. The total plasmatic concentration of free amino acids in AH-130-bearing rats was decreased at Day 4, when the tumor was actively proliferating, and returned to control values at Day 10, when the tumor had reached a stationary state. On the contrary, in pair-fed animals total plasma amino acids decreased over the whole experimental period. Plasma branched-chain amino acids were unchanged or even decreased in tumor hosts, while the Gly/Pro ratio was elevated in pair-fed rats. The intracellular concentration of free amino acids was higher in stationary than in exponentially- growing tumors, reflecting the enhanced proteolytic rates observed in stationary tumor cells. On the whole, the results suggest that reduced food uptake and metabolic competition by the tumor are not sufficient to justify the marked hypercatabolism in host tissues during the AH-130 hepatoma growth. The profound differences between tumour-bearing and pair-fed animals suggest that, if malnutrition undoubtedly played a role in this model of cancer cachexia, its effects were overwhelmed and subverted in the frame of the tumour-host interplay that dictated a distinctly peculiar syndrome.

A flow cytometric study of the rat Yoshida AH-130 ascites hepatoma

The implantation of the Yoshida AH-130 ascites hepatoma to rats results in a marked reduction in body weight in the tumour-bearing hosts. This is associated with an important reduction in both food intake and energetic efficiency in the last period of tumour growth. The growth of the tumour mass has a clear initial exponential phase, in which the maximum cell density is reached, with the majority of cells being in the synthetic phase (S) followed by most of the cells being in the G 0 G 1 phase, as determined using flow cytometry.

Cell death by apoptosis in the Yoshida ascites hepatoma AH-130

Cell death by apoptosis in the Yoshida ascites hepatoma AH-130

Effects of systemic inhibition of prostaglandin production on protein metabolism in tumor-bearing rats.

Two rat tumors, Morris hepatoma 7777 (MH) and Yoshida ascites hepatoma AH130 (YAH) were compared, and the influence of systemic inhibition of prostaglandin (PG) synthesis on muscle protein metabolism was evaluated. Tumor-bearing rats were compared with ad libitum- and pair-fed controls. Rats were also treated with naproxen, an inhibitor of PG synthesis. Tumors caused progressive anorexia and weight loss and resulted in decreased weight and/or protein content of the soleus, extensor digitorum longus, and epitrochlearis muscles. The extent of this wasting varied with muscle and tumor type. Muscle wasting induced by the tumors appeared to result from increased protein degradation and/or decreased protein synthesis, as determined in isolated epitrochlearis muscle. In YAH, reduced feed intake did not appear to be responsible for muscle wasting; however, in MH, it accounted for a significant proportion of the muscle loss. YAH produced large amounts of PGE2. Treatment of rats with naproxen inhibited tumor PGE2 production and muscle protein loss in rats bearing YAH. Naproxen had no effect on muscle weight or protein degradation in rats bearing MH. These results would appear to implicate PGE2 in the development of cachexia in the laboratory rat.

In vivo activity on murine tumors of a novel antitumor compound, N-beta-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]phenazine-6-carboxamide sodium salt (NC-190).

A novel antitumor compound, N-beta-dimethyl-aminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]-phenazine-6-carboxamide sodium salt (NC-190) was evaluated for its antitumor activity in experimental murine tumor systems. In the initial studies with P388 leukemia (i.p.-i.p.), NC-190 led to an increase of greater than 200% in life span (ILS), and 75% of the mice were alive on day 30, when the optimal dose (50 mg/kg, days 1-5) was given. Additionally, the compound had significant activities against i.p. inoculated mouse L1210 leukemia, B16 melanoma, M5076 reticulum cell sarcoma, sarcoma 180, mouse hepatoma MH134, and rat Yoshida sarcoma and Yoshida ascites hepatoma AH130. The optimal dose resulted in a greater than 280% ILS with a 30-day survival of 50% in mice with L1210 leukemia (100 mg/kg, days 1-5), a 156% ILS in mice with B16 melanoma (50 mg/kg, days 1-5), a 98% ILS with a 90-day survival of 25% in mice with M5076 reticulum cell sarcoma (25 mg/kg, days 1, 5, 9, and 13), a greater than 300% ILS with a 60-day survival of 50% in mice with sarcoma 180 (50 mg/kg, days 3-10), a 148% ILS with a 60-day survival of 25% in mice with MH134 (25 mg/kg, days 1-5), a 129% ILS with a 60-day survival of 12.5% in rats with Yoshida sarcoma (12.5 mg/kg, day 3-10), and a greater than 161% ILS with a 60-day survival of 50% in rats with AH130 (6.3 mg/kg, days 3-10). In the experiments with s.c. inoculated tumors, NC-190 not only inhibited tumor growth, but also increased the life span of mice with Lewis lung carcinoma or B16 melanoma. The 60-day survivors accounted for 60% and 30% in mice with Lewis lung carcinoma and B16 melanoma, respectively. The compound significantly inhibited the spontaneous lung metastasis of Lewis lung carcinoma by more than 90% when eight daily i.v. injections were given. NC-190 was active by the i.p., s.c., and i.v. routes. Five consecutive daily i.p. doses (days 1-5) were more effective than a single dose (day 1), two doses (days 1 and 5), or three doses (days 1, 5, and 9). NC-190 warrants further study as a potential antineoplastic agent against human neoplasms, as it has a broad spectrum of antitumor activity and inhibits metastasis.

Effect of two aliphatic aldehydes, methylglyoxal and 4-hydroxypentenal, on the growth of Yoshida ascites hepatoma AH-130

The influence of a ketoaldehyde, methylglyoxal (MG), and a hydroxyalkenal, 4-hydroxypentenal (HPE), on the growth of a highly-deviated tumour has been investigated. MG and HPE, administered intraperitoneally, strongly depressed in rats the proliferative activity of the Yoshida ascites hepatoma AH-130, reducing its mitotic and labelling indices as well as the proportion of cycling cells (growth fraction). Monitoring the effects on the cell cycle by the labelled mitoses method showed that the percentage of labelled mitoses was markedly lowered after either aldehyde, which is indicative for a blocking effect in the S phase. In addition, the mean cell cycle time was slightly prolonged by MG, probably due to accumulation of cells in G1, whereas HPE delayed the first mitotic peak and increased the mean DNA synthetic period without modifying the overall cycle time. The effects of HPE on the cell cycle were prevented by pretreatment with polyamines. Repeated doses of MG significantly increased the fraction of tumour-bearing rats surviving at 90 days (‘indefinite’ survivors) as well as the survival time of those which succumbed, implying that the carcinostatic effect of MG persisted over several cell cycles. By contrast, HPE did not significantly modify the survival of AH-130-bearing rats, suggesting that its influence on tumour growth was rapidly reversible.

Regulation of protein turnover versus growth state

Yoshida AH-130 ascites hepatoma cells were grown in rats and were examined by quantitative electron microscopy 4 days (exponential growth phase) and 10 days (stationary phase) after intraperitoneal inoculation. No significant differences between growing and growth-inhibited tumors were found in the composition of the cytoplasm, except for a slight increase in the volume fraction of mitochondria (from 9.6 to 12.1%) and, in particular, a prominent (4.2-fold) increase in the volume fraction of early stages of autophagic vacuoles (from 0.31 × 10-4 at day 4 to 1.37× 10-4 at day 10;P 0.001). At the same time, the rate of cell protein degradation was increased twofold, namely from 0.67%/h at day 4 to 1.37%/h at day 10, as measured in vitro after labeling cells with 3H-leucine in vivo. Such elevated proteolytic activity was entirely suppressed by ammonia, which inhibits the lysosomal pathway for protein degradation. The data show that: (i) the regulation of autophagic degradation of cytoplasmic constituents depending on the growth state was maintained in these tumor cells, and (ii) the increase in autophagy measured by morphometric analysis contributed to, yet did not quantitatively explain, the acceleration of protein degradation characterizing the transition from the logarithmic growth phase to the growth-inhibited state.

Regulation of protein turnover versus growth state. Studies on the mechanism(s) of initiation of acidic vacuolar proteolysis in cells of stationary ascites hepatoma.

1. After transplantation, the rat AH-130 Yoshida ascites hepatoma enters a phase of exponential (log) growth, followed by a quasi-stationary (sta) state. Combining measurements made in vivo and in vitro, cessation of protein accumulation (growth) in sta phase has previously been shown to result from convergent reduction of protein synthesis and enhancement of protein breakdown [Tessitore, Bonelli, Cecchini, Amenta & Baccino (1987) Arch. Biochem. Biophys. 255, 372-384]. 2. One day after labelling in the animal with [3H]leucine, AH-130 cells were processed for short-term assays in vitro to measure rates of endogenous protein breakdown. 3. Exposure of AH-130 cells to inhibitors interfering with different steps of the acidic vacuolar pathway (AVP) showed that: (i) in log tumour cells the AVP was extensively suppressed; (ii) in sta tumour cells virtually all of the proteolytic acceleration was accounted for by activation of the AVP. 4. Treating log tumour cells with glucagon, cyclic AMP, or nutritional deprivation failed to elevate substantially the proteolytic rates. Nor could the elevation in proteolysis be explained by changes in free amino acids, which were more concentrated in the ascitic fluid of sta tumours. 5. The enhanced proteolysis in sta tumour cells was not associated with any increase in the intracellular activity levels of lysosomal cathepsins B, D, H, and L. 6. The above growth-related modulation of protein breakdown in AH-130 cells was probably a reflection of the tumour growth state rather than the direct effect of environmental stimuli.

Studies on ornithine decarboxylase from liver, kidney and tumoral tissues during activity decay following cycloheximide administration

We have studied the activity, thiol-dependency and K m of ornithine decarboxlase (ODC) from the following sources: liver of rats subjected to partial hepatectomy or administered thioacetamide, the rat 3924A Morris hepatoma, the rat AH130 Yoshida ascites hepatoma, a mouse transplantable mammary carcinoma and kidney of rats administered testosterone propionate. In order to detect possible changes occurring during in vivo ageing of this enzyme we inhibited protein synthesis with cycloheximide. A gradual decrease of K m during ageing was observed in ODC from liver.

Heterogeneous regulation of lysosomal hydrolase activities in rat liver and Yoshida ascites hepatoma AH-130

Heterogeneous regulation of lysosomal hydrolase activities in rat liver and Yoshida ascites hepatoma AH-130

Control of protein degradation and growth phase in normal and neoplastic cells.

Cells have to double their protein mass in order to divide. Whether this is achieved through increased synthesis (PS), decreased degradation (PD), or a combination of both is still debated. Likewise open are other basic questions: whether, beyond differences relating to growth phase (GP) or rate, reduced PD rates are a general characteristic of neoplastic versus normal cells, conferring to them a definite growth advantage; which mechanisms are operating the PD regulation, if any, during GP transitions, and which ones may be defective in neoplastic cells. Growing liver under conditions of regeneration or development is known to achieve a net protein accumulation thanks to increased PS, and particularly, to decreased PD rates, as compared with the adult, steady-state tissue; the level of lysosomal proteinase (LP) activities is reduced; in the regenerating liver this reduction has been located in cycling hepatocytes. AH-130 Yoshida ascites hepatoma cells effect the transition from log to stationary GP by concurrently reducing PS and accelerating PD (slow turnover protein pool); while PD is virtually not affected by lysosomal inhibitors (LI) in growing cells, the extra PD in resting cells is all inhibitable; there is no regulation of LP levels over this GP transition, which is due to depletion of oxygen and nutrients. GP transitions in normal 3T3 cells are also coupled with regulations of both PS and PD, the extra PD in quiescent cells being all suppressible by LI. Quiescence of 3T3 cells, due to depletion of growth factors, is associated with a marked elevation of some LP activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of cDNA sequences for rat M2-type pyruvate kinase and regulation of its mRNA.

Rat M2-type pyruvate kinase mRNA was enriched from total polysomes isolated from AH-130 Yoshida ascites hepatoma cells, which contain a very high concentration of the M2-type enzyme, by immunoprecipitation with a specific antibody and Staphylococcus aureus cells. Double-stranded cDNA synthesized from the enriched mRNA was inserted into the PstI site of pBR322, and the resultant recombinant DNA molecules were used to transform Escherichia coli. Three clones containing DNA complementary to M2-type pyruvate kinase mRNA were identified by colony hybridization, hybrid-arrested translation, and hybrid-selected translation. A partial restriction map was constructed covering about 1.44 kilobase pairs. The cloned region of the M2-type mRNA showed a high degree of sequence homology with the M1-type mRNA and some homology with the L-type mRNA as determined by dot blot hybridization. The molecular size of the M2-type mRNA, which was estimated to be 2.35-2.65 kilobases on denaturing gel, was the same as that of the M1-type mRNA. The level of hepatic M2-type pyruvate kinase mRNA measured by hybridization assay using cloned cDNA as a probe was increased 2.5-fold 1 day and 3.9-fold 2 days after partial hepatectomy and then started to decrease. This induction was followed by similar changes in the enzyme activity. AH-130 hepatoma cells contained 100-150 times more M2-type isozyme mRNA than regenerating liver. These results suggest that the increased levels of M2-type isozyme in regenerating liver and hepatoma cells are primarily due to elevation of hybridizable M2-type mRNA concentration.

The M1 and M2 subunits of rat pyruvate kinase are encoded by different messenger RNAs.

The question of whether the M1- and M2-type isozymes of pyruvate kinase are synthesized by a common mRNA was examined by isolating total RNA from rat skeletal muscle and AH-130 Yoshida ascites hepatoma cells, which express the M1- and M2-type isozymes, respectively, and translating the preparations in a rabbit reticulocyte lysate system. The newly synthesized pyruvate kinase subunits were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing under the denaturing condition. The pyruvate kinase subunit synthesized from hepatoma cell RNA had a slightly larger molecular weight and higher pI value than the subunit from muscle RNA. When a mixture of the two RNAs was added to the lysate, the same result was obtained. Thus, it is concluded that the M1- and M2-type isozymes are translated from different messenger RNAs.

Isolation and characterization of the plasma membrane from Yoshida hepatoma cells

Plasma membranes isolated from Yoshida ascites hepatoma AH-130 by a modification of the method of T.K. Ray (Biochim. Biophys. Acta 196:1, 1970), were subfractionated into three fractions having densities (d) 1.12, 1.14 and 1.16 by discontinuous sucrose density-gradient. Membrane subfractions were characterized by electron-microscopy, by assay of marker enzymes and by lipid composition. All subfractions appeared to be essentially free from whole mitochondria, lysosomes and nuclei. Subfraction d 1.16 had the highest 5'-nucleotidase, Mg++-ATPase and (Na+ +K+)-ATPase activities; cytochrome c oxidase was undetectable in any fraction and glucose-6-phosphatase was measurable only in fraction d 1.14 and 1.16. Cyclic AMP phosphodiesterase was nearly equally distributed in the fractions. Adenylate cyclase, 5'-nucleotidase and Mg++-ATPase activities of tumor membrane were lower with respect to liver plasma membrane, while cyclic AMP phosphodiesterase and (Na" +K+)-ATPase were found to have similar activities in the two membrane preparations. With respect to liver membrane, hepatoma membrane contained a higher amount of glycolipids and a higher amount of phospholipids accounted for mainly by sphingomyelin, phosphatidylserine and phosphatidic acid. The possible significance of the decrease of adenylate activity in the hepatoma membrane is briefly discussed.

Acceptor control ratio of mitochondria: Factors affecting it in morris hepatoma 5123 and yoshida hepatoma AH-130

Mitochondria from AH-130 Yoshida ascites hepatoma and 5123 Morris hepatoma show high acceptor control and ADP:O ratios approaching the theoretical values when suspended in a reaction mixture containing EDTA. Bovine semm albumin or NaF do not completely substitute for EDTA in achieving high acceptor control. The ATPase activity of these mitochondria is stimulated by Mg 2+; this metal ton also has harmful effect on the acceptor control and the ADP:O ratios. EDTA induces a strong inhibition of the ATPase basal activity (unstimulated by dinitrophenol oi added Mg2+) Hepatoma mitochondria, incubated in the presence of EDTA, respond well to different regulatory mechanism such as changes in mitochondrial concentration, phosphate potential, and endogenous substrates content. The phosphorylative apparatus of hepatoma mitochondria exhibits, even in the presence of protective agents, a higher lability than that of mitochondria from neonatal and adult rat livers. It is suggested that, although a slight degree of functional and perhaps structural damage occurs in hepaloma mitochondria in vitro, it is possible to obtain a high degree of coupling if protective agents are added to the suspensions and appropriate experimental conditions are selected.

Effect of chemical, physical and enzymic treatments on lysosomes from AH-130 yoshida ascites hepatoma.

Omogenati e frazioni L di epatoma ascite di Yoshida AH-130 sono piu resistenti di quelli di fegato all'attivazione termica a pH 5, al trattamento ipotonico ed alla digestione con tripsina.

Effect of trypsin on morphological integrity and some functional activities of mitochondria from normal liver and AH-130 Yoshida ascites hepatoma

The effect of trypsin on morphological integrity and certain functional activities of mitochondria from normal liver and AH-130 Yoshida ascites hepatoma, has been studied. Liver mitochondria are barely attacked by trypsin which induces swelling only id added in large amounts. The swelling is preceeded by a lag phase during which liberation of little amounts of ninhydrin reacting compounds occurs. Tumour mitochondria are highly sensitive to trypsin, which induces immediate swelling even if added in amounts as little as 0.9 μg/mg protein. Evidence is provided that in tumour mitochondria trypsin induces damage of inner membrane permselectivity towards sucrose, and respiratory inhibition, also when it fails to inactivate the external membrane enzyme, NADH:cytochrome c reductase. The results are interpreted as indicative of the existence of a different structural state in membranes of normal and tumour mitochondria.