Ascites Hepatoma Research Articles

THE COMPARATIVE MORPHOLOGICAL ANALYSIS OF THE NEPHROTOXICITY OF DOXORUBICIN AND NANOSTRUCTURED DOXORUBICIN IN CONDITIONS OF TRANSPLANTABLE CARCINOGENESIS

The study was carried out on 120 mongrel white laboratory rats, to whom were transplanted the ascitiс Zaidel's hepatoma and which were divided into three equal groups. Of these, the first group of animals was a control group without treatment. In the second group, to animals doxorubicin was injected, in the third group, nanostructured doxorubicin was injected to the animals for 21 days. The aim of the study was to assess the degree of morphological changes in the tubules and glomeruli of the kidneys under the influence of nanostructured doxorubicin and doxorubicin in the condition of transplantable carcinogenesis. Given the nephrotoxicity of the chemotherapy drugs studied, the dynamics of structural changes in the tubules and glomeruli of the kidneys was studied. Conducted a comparative morphological evaluation of the changes occurring in the kidneys. The results of a morphological study of the structural components of the kidneys prove that doxorubicin and nanostructured doxorubicin have different effects on both the tubular apparatus and the glomeruli of the kidneys in the condition of transient carcinogenesis. The use of doxorubicin is accompanied by a relatively pronounced nephrotoxic effect, which is indicated by dystrophic and necrobiotic changes in the epithelium of convoluted tubules and the glomerular apparatus of the kidneys. The use of nanostructured doxorubicin is limited to moderately expressed dystrophic changes in the epithelium of the tubular apparatus of the kidneys, the glomerular apparatus remains intact at the same time. Thus, the use of the preparation of nanostructured doxorubicin for the treatment of the ascitic hepatoma of Zaidel in experimental animals has a less pronounced toxic and damaging effect on the renal parenchyma.

Respiratory complex II in mitochondrial dysfunction-mediated cytotoxicity: Insight from cadmium

In the present work we studied action of several inhibitors of respiratory complex II (CII) of mitochondrial electron transport chain, namely malonate and thenoyltrifluoroacetone (TTFA) on Cd2+-induced toxicity and cell mortality, using two rat cell lines, pheochromocytoma PC12 and ascites hepatoma AS-30D and isolated rat liver mitochondria (RLM). It was shown that malonate, an endogenous competitive inhibitor of dicarboxylate-binding site of CII, restored in part RLM respiratory function disturbed by Cd2+. In particular, malonate increased both phosphorylating and maximally uncoupled respiration rates in KCl medium in the presence of CI substrates as well as palliated changes in basal and resting state respiration rates produced by the heavy metal on the mitochondria energized by CI or CII substrates. Notably, malonate enhanced Cd2+-induced swelling of the mitochondria energized by CI substrates in KCl and, in a much lesser extent and at higher [Cd2+], in sucrose media but did not influence on the Cd2+ effects in NaCl medium. Besides, malonate did not affect swelling in sucrose media of RLM energized by CIV substrates under using of Cd2+ or Ca2+ whereas it strongly increased the mitochondrial swelling produced by selenite. In addition, malonate produced some protection against Cd2+-promoted necrotic death of AS-30D and PC12 cells and reduced intracellular reactive oxygen species (ROS) formation evoked by Cd2+ in PC12 cells. Importantly, TTFA, an irreversible competitive inhibitor of Q-binding site of CII, per se induced apoptosis of AS-30D cells which was inhibited by co-treatment with Cd2+ as well as decreased the Cd2+-enhanced intracellular ROS formation. In turn, decylubiquinone (dUb) at low μM concentrations did not protect AS-30D cells against the Cd2+-induced necrosis and enhanced the Cd2+-induced apoptosis of the cells. High μM concentrations of dUb were highly toxic for the cells. As consequence, the findings give new evidence indicative of critical involvement of CII in mechanism(s) of Cd2+-produced cytotoxicity and support the notion on CII as a perspective pharmacological target in mitochondria dysfunction-mediated conditions and diseases.

Effects of the beta2 agonist formoterol on atrophy signaling, autophagy, and muscle phenotype in respiratory and limb muscles of rats with cancer-induced cachexia

Muscle mass loss and wasting are characteristic features of patients with chronic conditions including cancer. Beta-adrenoceptors attenuate muscle wasting. We hypothesized that specific muscle atrophy signaling pathways and altered metabolism may be attenuated in cancer cachectic animals receiving treatment with the beta2 agonist formoterol. In diaphragm and gastrocnemius of tumor-bearing rats (intraperitoneal inoculum, 108 AH-130 Yoshida ascites hepatoma cells, 7-day study period) with and without treatment with formoterol (0.3 mg/kg body weight/day/7days, subcutaneous), atrophy signaling pathways (NF-κB, MAPK, FoxO), proteolytic markers (ligases, proteasome, ubiquitination), autophagy markers (p62, beclin-1, LC3), myostatin, apoptosis, muscle metabolism markers, and muscle structure features were analyzed (immunoblotting, immunohistochemistry). In diaphragm and gastrocnemius of cancer cachectic rats, fiber sizes were reduced, levels of structural alterations, atrophy signaling pathways, proteasome content, protein ubiquitination, autophagy, and myostatin were increased, while those of regenerative and metabolic markers (myoD, mTOR, AKT, and PGC-1alpha) were decreased. Formoterol treatment attenuated such alterations in both muscles. Muscle wasting in this rat model of cancer-induced cachexia was characterized by induction of significant structural alterations, atrophy signaling pathways, proteasome activity, apoptotic and autophagy markers, and myostatin, along with a significant decline in the expression of muscle regenerative and metabolic markers. Treatment of the cachectic rats with formoterol partly attenuated the structural alterations and atrophy signaling, while improving other molecular perturbations similarly in both respiratory and limb muscles. The results reported in this study have relevant therapeutic implications as they showed beneficial effects of the beta2 agonist formoterol in the cachectic muscles through several key biological pathways.

Fiber‐type specificity of cancer cachexia‐induced muscle wasting is phosphorylated p70S6K‐dependent

The purpose of the present study is to investigate the differences between slow‐ and fast‐twitch muscles in cancer cachexia‐induced muscle wasting.Twelve male Wistar rats were randomly divided into two groups; control and cancer cachexia‐induced group. The cancer cachexia‐induced rats received an intraperitoneal injection of AH‐130 ascites hepatoma cells. The serum, the soleus and plantaris muscles were collected nine days after injection. Then, the expression levels of TNF‐α, p70S6K, FoxO1, FoxO3a, IL‐6, Atrogin‐1, and MuRF‐1 were measured.The cachexia‐induced rats resulted in a loss in body weight and muscle mass, and an increase in the expression level of TNF‐α in serum. In addition, the mass in fast ‐twitch muscle decreased significantly higher than that in slow‐twitch muscle. The levels of Atrogin‐1 and MuRF‐1 expression, which are the indicators related to protein degradation, increased in the cachexia‐induced group compared with the control group. However, there were no significant differences in Atrogin‐1 and MuRF‐1expression between slow‐ and fast‐twitch muscles. The expression level of phosphorylated p70S6K, which is one of the indicators related to protein synthesis, decreased in the cachexia‐induced group. In addition, the expression level of phosphorylated p70S6K in the cachexia‐induced group was significantly lower in fast‐twitch than in slow‐twitch muscles.These results indicated that cancer cachexia‐induced both in slow‐ and fast‐twitch muscle wasting might be caused by increased protein degradation factors whereas preferential fast‐twitch muscle wasting depended on the suppression of phosphorylated p70S6K expression, which was different from slow muscle wasting.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

On reprogramming of tumor cells metabolism: detection of glycogen in the cell lines of hepatocellular origin with various degrees of dedifferentiation.

The reprogramming of cancer cells includes shifts in glucose and glycogen metabolism. The aim of our work was to check the ability of forming glycogen grains in hepatocellular tumor cell lines of various dedifferentiation levels. We studied the monolayer culture established in vitro after explanting cells from rat ascites Zajdela hepatoma strain C (ZH-C) as a "parental" line and its five daughter clonal sublines: the holoclonal sublines 3H, 5F, 6H and the meroclonal ones 1E, 9C, which possess, respectively, the properties of cancer stem-like cells (CSLCs) and cancer progenitor-like cells(CPLCs). Besides, we studied four permanent cell lines of a rat hepatoma HTC, two murine hepatomas BWTG3 and MH-22a,and human hepatoblastoma HepG2. We used normal rat hepatocytes as positive control cells that form glycogen. We estimated relative cell dedifferentiation levels of the studied lines via analysis of cell morphology, morphometry and motility character on stained cell preparations and lifetime video files. Glycogen in the cells was detected using a Schiff type Au-SO2 reagent. All studied hepatocellular tumor lines were not of equal dedifferentiation level as manifested by different nucleus-to-cytoplasm ratio, by epithelium-like or fibroblast-like morphology, by tight or loosen intercellular contacts, by cell migration of collective or individual types. Glycogen fluorescence of uneven intensity was observed in all normal rat hepatocytes, but only in some cell groups or in single cells of hepatocellular tumor lines. The large or small fluorescent grains were found not only in relatively less dedifferentiated parental ZH-C line, BWTG3 and HepG2 lines, but also in moderately dedifferentiated 1E and HTC lines and even in severely dedifferentiated 3H, 5F and 6H sublines, as well as in the islets of the rat ascites hepatoma induced in vivo by the injection of 3H cells (the tumor-initiating cells). On the other hand, MH-22 and 9C lines, being relatively less and moderately dedifferentiated, showed no glycogen fluorescence. Thus,in 10 tumor cell lines of hepatocellular origin, an ability to reserve glycogen manifested no obvious dependency on their dedifferentiation level. Glycogen grains were detected in some cells even of the severely dedifferentiated lines: in single CSLCs of holoclonal ZH sublines grown in vitro and in a majority of tumor-initiating cells derived from ascites hepatoma in vivo. We suggest that dynamic changes in glycogen formation in CSLCs and tumor-initiating cells might be of importance for their dedifferentiation, self-renewal in vitro, survival and metastasis in vivo. The role of glycogen in maintaining viability and metastasis of tumor cells is to be further studied.

Formoterol attenuates increased oxidative stress and myosin protein loss in respiratory and limb muscles of cancer cachectic rats.

Muscle mass loss and wasting are characteristic features of patients with chronic conditions including cancer. Therapeutic options are still scarce. We hypothesized that cachexia-induced muscle oxidative stress may be attenuated in response to treatment with beta2-adrenoceptor-selective agonist formoterol in rats. In diaphragm and gastrocnemius of tumor-bearing rats (108 AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally) with and without treatment with formoterol (0.3 mg/kg body weight/day for seven days, daily subcutaneous injection), redox balance (protein oxidation and nitration and antioxidants) and muscle proteins (1-dimensional immunoblots), carbonylated proteins (2-dimensional immunoblots), inflammatory cells (immunohistochemistry), and mitochondrial respiratory chain (MRC) complex activities were explored. In the gastrocnemius, but not the diaphragm, of cancer cachectic rats compared to the controls, protein oxidation and nitration levels were increased, several functional and structural proteins were carbonylated, and in both study muscles, myosin content was reduced, inflammatory cell counts were greater, while no significant differences were seen in MRC complex activities (I, II, and IV). Treatment of cachectic rats with formoterol attenuated all the events in both respiratory and limb muscles. In this in vivo model of cancer-cachectic rats, the diaphragm is more resistant to oxidative stress. Formoterol treatment attenuated the rise in oxidative stress in the limb muscles, inflammatory cell infiltration, and the loss of myosin content seen in both study muscles, whereas no effects were observed in the MRC complex activities. These findings have therapeutic implications as they demonstrate beneficial effects of the beta2 agonist through decreased protein oxidation and inflammation in cachectic muscles, especially the gastrocnemius.

ANTITUMOR EFFECT OF HYDROGEL CISPLATIN ON ZEJDEL ASCITES HEPATOMA

Aims and objectives. During regional intraperitoneal chemotherapy cytostatics in prolonged dosage form, in particular, on the basis of various biodegradable hydrogels, such as dextran phosphate are applied. Aim of the study - a comparative evaluation of the antitumor activity ofcisplatin and cisplatin in prolonged dextran phosphate hydrogel form on Zajdel ascites hepatoma. Materials and methods. On 49 white no inbreed rats (n = 7) with implanted intraperitoneal Zajdel ascites hepatoma the antitumor activity of cisplatin in prolonged dextran phosphate hydrogel form with single intraperitoneal injection of the dosage from 3 to 8 mg/kg (≈ maximum tolerated dose, MTD) was evaluated in comparison with officinal cisplatin. The treatment efficacy was assessed by a statistically significant standard criteria: the absence of ascites by 32nd day of the experiment (complete remission) and an increase in life span of rats with ascites (T/C >_125 %, «treatment/control») in comparison with the rats without treatment (tumor growth control, where T/C = 100 %) or treated with officinal cisplatin. Macroscopic, pathomorphological and statistical analysis of the results were used. Results. It has been shown that hydrogel form of cisplatin compared to cisplatin was significantly more effective: complete remission 4/7-6/7 vs 0/7, T/C = 140-188 % vs 91-101 %, the volume of ascites 13,6 ± 6,6 ml vs 50,0 ± 7,9 ml (p = 0,004) because of a better tolerance. Regression analysis confirms that hydrogel form of cisplatin in used dose range significantly improves the rats survival with Zajdel ascites hepatoma, reducing the risk of death from tumor in 7-35 times and the relative risk with the dose of 5,5 mg/kg in 36 times (95 % CI3,86 + 327,60) (p = 0,002-0,005). Conclusions. The data obtained allow considering the hydrogel form of cisplatin as a promising prolonged this drug form of cisplatin by the intraperitoneal therapy and recommending it to preclinical study.

Effect of the autonomic nervous system on cancer progression depends on the type of tumor: solid are more affected then ascitic tumors.

A number of recently published studies have shown that the sympathetic nervous system may influence cancer progression. There are, however, some ambiguities about the role of the parasympathetic nerves in the modulation of growth of different tumor types. Moreover, tumor models used for investigation of the autonomic neurotransmission role in the processes related to the cancer growth and progression are mainly of the solid nature. The knowledge about the nervous system involvement in the modulation of the development and progression of malignant ascites is only fragmental. Therefore, the aim of the present article was to summarize the results of our experimental studies focused on the elucidation of the role of the autonomic nervous system in the modulation of tumor growth in animals. We are summarizing data from studies, in which not only different experimental approaches in order to influence the autonomic neurotransmission, but also different tumor models have been used. Three different types of tumor models, namely solid rat intra-abdominal fibrosarcoma, solid murine subcutaneous melanoma, and rat ascites hepatoma, and three types of interventions have been used in order to modulate the autonomic neurotransmission, specifically chemical sympathectomy, subdiaphragmatic vagotomy, or the electric stimulation of the vagus nerve. We have proved a strong stimulatory effect of the sympathetic nerves on the development and growth in both solid tumors, rat fibrosarcoma as well as murine melanoma, and significant inhibitory impact on the survival time of tumor-bearing animals. The progression of ascites hepatoma in rats was not influenced by chemical sympathectomy. Modulation of parasympathetic signalization by vagotomy or vagal nerve stimulation does not affect fibrosarcoma and ascites hepatoma growth and survival of the tumor-bearing rats. Based on the obtained data, it seems that the solid types of tumors are suitable substrate for the direct action of neurotransmitters released especially from the sympathetic nerves. In contrast, it appears that the malignant ascites are not under the direct autonomic nerves control; however, an indirect action via the immune functions modulation cannot be excluded.

Inhibition of Non-flux-Controlling Enzymes Deters Cancer Glycolysis by Accumulation of Regulatory Metabolites of Controlling Steps.

Glycolysis provides precursors for the synthesis of macromolecules and may contribute to the ATP supply required for the constant and accelerated cellular duplication in cancer cells. In consequence, inhibition of glycolysis has been reiteratively considered as an anti-cancer therapeutic option. In previous studies, kinetic modeling of glycolysis in cancer cells allowed the identification of the main steps that control the glycolytic flux: glucose transporter, hexokinase (HK), hexose phosphate isomerase (HPI), and glycogen degradation in human cervix HeLa cancer cells and rat AS-30D ascites hepatocarcinoma. It was also previously experimentally determined that simultaneous inhibition of the non-controlling enzymes lactate dehydrogenase (LDH), pyruvate kinase (PYK), and enolase (ENO) brings about significant decrease in the glycolytic flux of cancer cells and accumulation of intermediate metabolites, mainly fructose-1,6-bisphosphate (Fru1,6BP), and dihydroxyacetone phosphate (DHAP), which are inhibitors of HK and HPI, respectively. Here it was found by kinetic modeling that inhibition of cancer glycolysis can be attained by blocking downstream non flux-controlling steps as long as Fru1,6BP and DHAP, regulatory metabolites of flux-controlling enzymes, are accumulated. Furthermore, experimental results and further modeling showed that oxamate and iodoacetate inhibitions of PYK, ENO, and glyceraldehyde3-phosphate dehydrogenase (GAPDH), but not of LDH and phosphoglycerate kinase, induced accumulation of Fru1,6BP and DHAP in AS-30D hepatoma cells. Indeed, PYK, ENO, and GAPDH exerted the highest control on the Fru1,6BP and DHAP concentrations. The high levels of these metabolites inhibited HK and HPI and led to glycolytic flux inhibition, ATP diminution, and accumulation of toxic methylglyoxal. Hence, the anticancer effects of downstream glycolytic inhibitors are very likely mediated by this mechanism. In parallel, it was also found that uncompetitive inhibition of the flux-controlling steps is a more potent mechanism than competitive and mixed-type inhibition to efficiently perturb cancer glycolysis.

Cancer cachexia-when proteasomal inhibition is not enough.

Cachexia is a life threatening syndrome associated with several diseases, such as end‐stage heart failure, end‐stage renal disease, chronic obstructive pulmonary disease, chronic inflammation (i.e. rheumatoid arthritis), acquired immune deficiency syndrome, and cancer.1, 2 Cachexia is found in 31–87% of cancer patients especially in advanced disease stages.3 It is characterized by progressive weight loss, metabolic alterations, fatigue, and persistent reduction of body cell mass in response to a malignant tumour.2, 4, 5, 6 The incidence of cachexia in cancer patients is dependent on the type and site of the tumour. While patients with non‐Hodgkin's lymphoma, breast cancer, and sarcomas show low incidences, rates up to 83% in pancreatic cancer patients, and over 85% in patients with gastric cancer have been found. Additionally, around 60% of small‐cell and non‐small‐cell lung cancer patients develop cachexia.7, 8, 9 Cancer cachexia affects the function of several organs such as muscle, adipose tissue, liver, brain, immune system, and heart, collectively decreasing patients' quality of life and worsening their prognosis. Therefore, cachexia must be considered as a true multi‐organ syndrome.10 Because cancer cachexia leads to a decrease in physical performance and quality of life,11 and is associated with poor survival (accounting for more than 20% of cancer deaths,7, 12, 13, 14, 15) it is of major clinical relevance. Even more so since cachectic patients show lower response rates to chemotherapy7 and a reduced tolerance to anticancer treatment.16 Despite its importance, weight loss in cancer patients is rarely recognized, assessed,17 or treated actively.18, 19 Thus, cancer cachexia represents an important underappreciated clinical syndrome.

Chronic Myeloid Leukemia and Hepatoblastoma: Two Cancer Models to Link Metabolism to Stem Cells.

Low oxygen tension is a critical aspect of the stem cell niche where stem cells are long-term maintained. In “physiologically hypoxic” stem cell niches, low oxygen tension restrains the clonal expansion of stem cells without blocking their cycling, thereby contributing substantially to favor their self-renewal. The capacity of stem cells, hematopoietic stem cells in particular, to reside in low oxygen is likely due to their specific metabolic profile. A strong drive to the characterization of this profile emerges from the notion that cancer stem cells (CSC), like normal stem cells, most likely rely on metabolic cues for the balance between self-renewal/maintenance and clonal expansion/differentiation. Accordingly, CSC homing to low oxygen stem cell niches is the best candidate mechanism to sustain the so-called minimal residual disease. Thus, the metabolic profile of CSC impacts long-term cancer response to therapy. On that basis, strategies to target CSC are intensely sought as a means to eradicate neoplastic diseases. Our “metabolic” approach to this challenge was based on two different experimental models: (A) the Yoshida’s ascites hepatoma AH130 cells, a highly homogeneous cancer cell population expressing stem cell features, used to identify, in CSC adapted to oxygen and/or nutrient shortage, metabolic features of potential therapeutic interest; (B) chronic myeloid leukemia, used to evaluate the impact of oxygen and/or nutrient shortage on the expression of an oncogenetic protein, the loss of which determines the refractoriness of CSC to oncogene-targeting therapies.

Effect of the specific proteasome inhibitor bortezomib on cancer-related muscle wasting.

BackgroundMuscle wasting, a prominent feature of cancer cachexia, is mainly caused by sustained protein hypercatabolism. The enhanced muscle protein degradation rates rely on the activity of different proteolytic systems, although the Adenosine triphosphate (ATP)‐ubiquitin‐proteasome‐dependent pathway and autophagy have been shown to play a pivotal role. Bortezomib is a potent reversible and selective proteasome and NF‐κB inhibitor approved for the clinical use, which has been shown to be effective in preventing muscle wasting in different catabolic conditions. The aim of the present study has been to investigate whether pharmacological inhibition of proteasome by bortezomib may prevent skeletal muscle wasting in experimental cancer cachexia.MethodsCancer cachexia was induced in rats by intraperitoneal injection of Yoshida AH‐130 ascites hepatoma cells and in mice by subcutaneous inoculation of C26 carcinoma cells. Animals were then further randomized to receive bortezomib. The AH‐130 hosts were weighted and sacrificed under anaesthesia, on Days 3, 4, 5, and 7 after tumour inoculation, while C26‐bearing mice were weighted and sacrificed under anaesthesia 12 days after tumour transplantation. NF‐κB and proteasome activation, MuRF1 and atrogin‐1 mRNA expression and beclin‐1 protein levels were evaluated in the gastrocnemius of controls and AH‐130 hosts.ResultsBortezomib administration in the AH‐130 hosts, although able to reduce proteasome and NF‐κB DNA‐binding activity in the skeletal muscle on Day 7 after tumour transplantation, did not prevent body weight loss and muscle wasting. In addition, bortezomib exerted a transient toxicity, as evidenced by the reduced food intake and by the increase in NF‐κB DNA‐binding activity in the AH‐130 hosts 3 days after tumour transplantation. Beclin‐1 protein levels were increased by bortezomib treatment in Day 3 controls but were unchanged on both Days 3 and 7 in the AH‐130 hosts, suggesting that an early compensatory induction of autophagy may exist in healthy but not in tumour‐bearing animals. Regarding C26‐bearing mice, bortezomib did not prevent as well body and muscle weight loss 12 days after tumour implantation.ConclusionsThe results obtained suggest that proteasome inhibition by bortezomib is not able to prevent muscle wasting in experimental cancer cachexia. Further studies are needed to address the issue whether a different dosage of bortezomib alone or in combination with other drugs modulating different molecular pathways may effectively prevent muscle wasting during cancer cachexia.

The effect of modulators of large-conductance Ca2+-modulated K+ channels on rat AS-30D ascites hepatoma cells and isolated liver mitochondria treated with Cd2+

ATP-producing cell organelles, mitochondria, are a primary target of heavy metals, major environmental pollutants causing a variety of diseases and pathologies. The mechanism of heavy metal toxic effect was established to include changes both in the intracellular production of reactive oxygen species (ROS) and mitochondrial dysfunction due to respiratory chain disorders and activation of the Ca2+-dependent non-selective pore in the mitochondrial inner membrane. Te role of other ion channels including such selective potassium channels as large-conductance Ca2+-activated K+ channels, BK(Ca), supposed to be cytoprotective, remains poorly studied. In the present work conducted on rat AS-30D ascites hepatoma cells and liver mitochondria, we studied the effect of different BK(Ca) effectors in the presence or absence of Cd2+ ions in the incubation medium, specifically of two activators, NS1619 and NS004, and one blocker, paxilline. After 24-h incubation of AS-30D cells with 10 μM of both NS1619 and NS004, the number of apoptized cells was found to increase significantly versus control; moreover, the presence of these BK(Ca) activators in the incubation medium exerted an additive effect on Cd2+-induced apoptosis of AS-30D cells. The same concentration of NS1619 and NS004 did not affect significantly AS-30D cellular respiration after 3, 24 and 48 h of incubation, although increasing after 3 h the intracellular production of ROS. In experiments on isolated rat liver mitochondria, NS1619 and NS004 added at the same concentration to the KCl-containing medium did not affect the rates of respiratory State 3 (after Chance) and maximally uncoupled respiration (both in the presence and absence of Cd2+); concurrently, they induced a weak uncoupling effect by increasing both basal and resting-state (State 3 after Chance) respirations, and also enhanced a high-amplitude mitochondrial swelling induced by Cd2+ in this medium. Paxilline (at 1μM) was shown to reduce the mortality of AS-30D cells after 3-, 24- and 48 h-incubation in the presence of Cd2+ and to increase the intracellular ROS production in control after 3 and 24 h of exposure. Paxilline added at a concentration exerting a long-term protective effect did not affect cellular respiration of rat AS-30D cells and isolated liver mitochondria (both in the presence and absence of Cd2+) and did not reduce mitochondrial swelling observed in the presence of Cd2+ and BK(Ca) activators. Possible molecular action mechanisms of BK(Ca) modulators are discussed.

Antitumor immunostimulatory activity of polysaccharides from Salvia chinensis Benth

Ethnopharmacological relevanceSalvia chinensis Benth (S. chinensis) is a traditional herb applied in the treatment of hepatocellular carcinoma (HCC). Polysaccharides abundantly exist in this plant. However, it remains poorly understood if polysaccharides from S. chinensis (PSSC) contribute to its anti-HCC activity. Materials and methodsThe in vivo anti-HCC activity of PSSC was evaluated in Kunming mice bearing H22 ascitic hepatoma cells. An array of physiological indexes was measured to evaluate toxicological effects on host animals. Subgroups of immune cells were purified by a magnetic-activated cell sorting system and analyzed by flow cytometry. Reverse transcription real-time PCR and immunoblotting were recruited to determine the effects of PSSC on the cellular signaling of different subgroup of immune cells. ResultsPSSC suppressed in vivo proliferation of H22 cells with undetectable toxic effects on tumor-bearing mice. PSSC alleviated tumor transplantation-induced CD4+ T cell apoptosis and dysregulation of serum cytokine profiles, which elevated cytotoxic activities of natural killer and CD8+ T cells. PSSC reduced serum levels of prostaglandin E2 (PGE2). Injection of exogenous PGE2 completely abrogated the antitumor immunostimulatory activity of PSSC. Cyclic adenosine monophosphate (cAMP) is the second messager of PGE2. In CD4+ T cells, PSSC substantially declined intracellular cAMP. This event elevated protein levels of JAK3, enhancing STAT5 phosphorylation and STAT5-depedent expression of anti-apoptotic genes. Cyclooxygenase-2 is the key enzyme mediating biosynthesis of PGE2. PSSC suppressed the transcription and translation of cyclooxygenase-2 in tumor associated macrophages. ConclusionOur data clearly showed antitumor immunostimulatory activity of PSSC against transplanted H22 HCC cells. Suppressing tumor transplantation-induced PGE2 production was implicated in the anti-tumor immunostimulatory activity of PSSC. These works provides novel insights into the traditional application of S. chinensis against HCC and supported considering PSSC as an adjuvant reagent in clinical HCC treatment.

Efficiency of Combining Free and Polymer-Immobilized Prospidin with Doxorubicin for Treatment of Zajdel Ascites Hepatoma in Rats

The antitumor activity and side effects of the drug combinations prospidin + doxorubicin and prospidin hydrogel + doxorubicin were studied in rats with transplanted Zajdel ascites hepatoma. Test animals were divided into one control group and five test groups depending on the therapy protocol. Control animals were not treated. Test groups received (i) an aqueous solution of prospidin; (ii) prospidin hydrogel; (iii) doxorubicin; (iv) prospidin + doxorubicin; and (v) prospidin hydrogel + doxorubicin. The maximum tolerated doses (MTDs) of the drugs were used in monotherapy; half the MTD, in combination chemotherapy. The antitumor activity was evaluated in terms of lethality, lethality from tumor progression, total cure rate, average life expectancy, and prolonged life expectancy. Side and toxic effects were evaluated using data on the blood cell composition and biochemical parameters. It was established that the antitumor activity of combined aqueous prospidin and doxorubicin did not exceed the activity of doxorubicin monotherapy. Side effects of this combination had the same character as those of doxorubicin and were represented by leuko- and thrombocytopenia and nephropathy. The antitumor effect of combined prospidin hydrogel + doxorubicin with respect to lethality was significantly more pronounced than the effect of each individual component. This combination had a statistically significantly lower toxicity than that of the most toxic component.

Partially isobaric peptide termini labeling assisted proteome quantitation based on MS and MS/MS signals.

The partially isobaric peptide termini labeling (PITL) method enabled to simultaneously obtain the quantitative information from MS and MS/MS spectrum, which combined the advantages of these two strategies. Relative quantification could be achieved by comparing the intensities of parent ions in MS spectra and a, b, y ions in the MS/MS spectra. The quantitative analysis for differently labeled yeast digests mixed at various ratios indicated the good accuracy, reproducibility, quantitative coverage and wide dynamic range of the PITL strategy. Finally, we found 84 differentially expressed proteins in mouse hepatocarcinoma ascites syngeneic cell lines with low and high lymph node metastasis rates with PITL strategy and 77 proteins of them were consistently quantified in our previous studies.

Эффективность комбинации свободной и полимер-связанной форм проспидина с доксорубицином у крыс с асцитной гепатомой Зайдела

Исследована противоопухолевая активность и побочные и токсические эффекты комбинаций проспидин + доксорубицин и проспидин — гидрогель + доксорубицин у крыс с трансплантированной асцитной гепатомой Зайдела. В зависимости от характера терапии животные были разделены на 6 групп (1 контрольную и 5 опытных). В контрольной группе животные лечения не получали, в опытных проводилась химиотерапия по следующим схемам: 1-я — проспидин, водный раствор, 2-я — проспидин-гидрогель; 3-я — доксорубицин; 4-я — проспидин + доксорубицин; 5-я — проспидин-гидрогель + доксорубицин при монохимиотерапии использовали максимально переносимые дозы, при комбинированной химитерапии — половинные от максимально переносимых. Для оценки противоопухолевой активности исследовали летальность, летальность от прогрессирования опухоли, частоту полного излечения; среднюю продолжительность жизни и увеличение продолжительности жизни. Для оценки побочных и токсических эффектов проводили исследование клеточного состава и биохимических показателей крови. Установлено, что при асцитной гепатоме Зайдела комбинация водного раствора проспидина с доксорубицином по противоопухолевой активности не превосходит монотерапию доксорубицином. Побочные эффекты данной комбинации соответствуют таковым у доксорубицина и представлены лейко- и тромбоцитопенией и нефропатией. Противоопухолевый эффект комбинации проспидин-гидрогеля с доксорубицином по влиянию на летальность достоверно более выражен, чем у каждого из ее компонентов. Комбинация отличается достоверно меньшей гематотоксичностью по сравнению с наиболее токсичным из ее составляющих.

Chemical sympathectomy increases neutrophil-to-lymphocyte ratio in tumor-bearing rats but does not influence cancer progression.

The sympathetic nervous system regulates many immune functions and modulates the anti-tumor immune defense response, too. Therefore, we studied the effect of 6-hydroxydopamine induced sympathectomy on selected hematological parameters and inflammatory markers in rats with Yoshida AH130 ascites hepatoma. We found that chemically sympathectomized tumor-bearing rats had significantly increased neutrophil-to-lymphocyte ratio, leukocyte-to-lymphocyte ratio, and plasma levels of tumor necrosis factor alpha. Although our findings showed that sympathetic denervation in tumor-bearing rats led to increased neutrophil-to-lymphocyte ratio, that is an indicator of the disease progression, we found no significant changes in tumor growth and survival of sympathectomized tumor-bearing rats.

Pattern RT1A and proteasome expression in cellular fractions of Zajdela ascitic hepatoma.

Pattern RT1A and proteasome expression in cellular fractions of Zajdela ascitic hepatoma.

Selenium-dependent antitumor immunomodulating activity of polysaccharides from roots of A. membranaceus

Roots of Astragalus membranaceus (Fish.) Bge. var. mongholicus (Bge.) Hsiao (A. membranaceus) have been long used as an auxiliary reagent supporting cancer treatment. Here, we compared the chemical composition and antitumor immunomodulating activity of polysaccharides from roots of A. membranaceus (PAMs) from five major habitats in Inner Mongolia, PR China. We revealed that compositions of monosaccharides and amino acids were comparable among PAMs from different habitats. However, amounts of selenium varied widely in roots of A. membranaceus and PAMs. PAMs selenium-dependently repressed the in vivo proliferation of transplanted H22 ascitic hepatoma and S180 sarcoma cells with low toxic impacts on tumor-bearing mice. Selenium-containing PAMs ameliorated host CD4+ T cell apoptosis and serum cytokine dysregulation induced by tumor transplantation, leading to the enhancement of cytotoxic activities of natural killer and CD8+ T cells. Moreover, PAMs also selenium-dependently improved the phagocytotic function of intra-abdominal macrophages and suppressed M2-like polarization of tumor-associated macrophages. These data suggested that the selenium content varies in the roots of A. membranaceus and PAMs from different geographical origins dramatically and selenium is an important contributor to the antitumor immunomodulation activities of PAMs.