Augmenter of liver regeneration potentiates doxorubicin anticancer efficacy by reducing the expression of ABCB1 and ABCG2 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is highly chemoresistant and therefore challenges both physicians and patients. Augmenter of liver regeneration (ALR), previously also known as 'hepatic stimulator substance', is reported to inhibit the epithelial-mesenchymal transition (EMT) in HCC, one of the frequent events that occur in cancer metastasis, suggesting that ALR is involved in HCC. In this study, we report for the first time that the transfection of ALR enhances the antitumor effect of chemotherapy with doxorubicin, a typical anticancer drug, on HCC in vitro and in vivo. The efflux of doxorubicin from ALR-transfected HCC cells is efficiently suppressed. This implies the intracellular retention of doxorubicin in tumor cells, which is at least partly attributable to the effective inhibition of ABCB1 and ABCG2 transporter expression in ALR-expressing cells. The downregulation of ALR expression by short hairpin RNA diminishes the antitumor effect of ALR. We further demonstrate that ALR inhibits the AKT/Snail signaling pathway, resulting in the downregulation of ABCB1 and ABCG2 expression. In conclusion, our results suggest that ALR is a potential chemotherapeutic agent against HCC.

Augmenter of liver regeneration ameliorates renal fibrosis in rats with obstructive nephropathy.

Renal fibrosis is a hallmark in CKD (chronic kidney disease) and is strongly correlated to the deterioration of renal function that is characterized by tubulointerstitial fibrosis, tubular atrophy, glomerulosclerosis and disruption of the normal architecture of the kidney. ALR (augmenter of liver regeneration) is a growth factor with biological functions similar to those of HGF (hepatocyte growth factor). In this study, our results indicate that endogenous ALR is involved in the pathological progression of renal fibrosis in UUO (unilateral ureteral obstruction) rat model. Moreover, we find that administration of rhALR (recombinant human ALR) significantly alleviates renal interstitial fibrosis and reduces renal-fibrosis-related proteins in UUO rats. Further investigation reveals that rhALR suppresses the up-regulated expression of TGF-ß1 (transforming growth factor ß1) induced by UUO operation in the obstructed kidney, and inhibits Smad2 and Smad3 phosphorylation activated by the UUO-induced injury in the animal model. Therefore we suggest that ALR is involved in the progression of renal fibrosis and administration of rhALR protects the kidney against renal fibrosis by inhibition of TGF-ß/Smad activity.

Augmenter of liver regeneration inhibits apoptosis of activated human peripheral blood lymphocytes in vitro.

Regulating apoptosis of lymphocytes is an effective strategy for treatment of lymphocyte-mediated diseases. Recently it has been demonstrated that augmenter of liver regeneration (ALR), an enigmatic protein presented ubiquitously in multiple forms among eukaryotes, possesses potent anti-apoptotic activity and supports proliferation of a variety of cells. However, its action on lymphocytes and the underlying mechanism are not completely understood. In this study, we analyzed the effects of recombinant human ALR (rhALR) on apoptosis of human lymphocytes activated with concanavalin A (ConA). Our results showed that rhALR inhibited apoptosis of ConA-activated lymphocytes and revealed reductions in the percentage of apoptotic cells, caspase-3 activation and PARP cleavage in cells treated with rhALR. Furthermore, the BAX/BCL-2 and cytosol/mitochondria cytochrome c ratios were decreased in the intrinsic death pathway and the activation of caspase-8 was also decreased in the extrinsic death pathway in activated lymphocytes treated with rhALR. In addition, rhALR significantly reduced the quantity of interleukin-2. These results demonstrated that rhALR has anti-apoptotic effects on activated lymphocytes through the activation of several apoptosis-related signaling pathways, and shed some light on the effects of rhALR on modulation immune reactions.

Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis.

Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-ß, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-ß and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner.

BACKGROUND/AIM: Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells. METHODS: Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [(3)H]-thymidine assay. RESULTS: The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1). CONCLUSION: rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.

Repression of cytochrome P450 activity in human hepatocytes in vitro by a novel hepatotrophic factor, augmenter of liver regeneration.

Pathological disorders of the liver were shown to be associated with an impairment of hepatic drug metabolism mediated in part by growth factors. Augmenter of liver regeneration (ALR) is a novel liver-specific hepatotrophic growth factor, whereas its action on cytochrome P450 (P450) metabolism is completely unknown. Application of ALR to primary human hepatocytes in vitro reduced P450 isoenzyme activities (1A2 and 2A6) in a dose-dependent manner. Time-course analysis revealed that the maximal inhibitory effect was reached after 24 to 72 h of exposure with 50 nM ALR. The reduction of basal activities upon ALR treatment was 35% for CYP1A2, 56% for CYP2A6, 18% for CYP2B6, and 45% for CYP2E1. Additionally, after induction of P450 with specific inducers, ALR revealed an inhibitory effect on the isoenzyme activities (CYP1A2, 41%; CYP2B6, 35%). Investigations of protein and mRNA expression of basal and induced CYP1A2 and CYP3A4 after ALR treatment by Western blotting and real-time reverse transcriptase-polymerase chain reaction, respectively, suggest a regulation on the transcriptional level. Furthermore, ALR treatment increased nuclear factor kB activity and reduced constitutive androstane receptor but not pregnane X receptor or aryl hydrocarbon receptor expression. In contrast, ALR revealed no effects on phase II reactions (glutathione/oxidized glutathione, UDP-glucuronyltransferase conjugation). Our results indicate that ALR, as a member of hepatotrophic factors, down-regulates basal and induced P450 in human liver and therefore cross-links growth signals to regulation of hepatic metabolism. These findings further imply a possible role of ALR in drug interactions during impaired hepatic function, whereas liver regeneration is triggered.

Further studies of sulfoxide-reducing enzyme system.

Various kinds of flavoenzymes such as NADPH-cytochrome c reductase, NADH-cytochrome b5 reductase, xanthine oxidase, lipoamide dehydrogenase and NADH dehydrogenase supplemented with their electron donors exhibited the sulfoxide reductase activity in the presence of a partially purified soluble factor from guinea pig liver. The present study suggests that new electron transfer systems in which the soluble factor functions as an electron carrier coupled with flavoenzymes described above are responsible for the sulfoxide reduction.

18-Hydroxylation of deoxycorticosterone by reconstituted systems from rat and bovine adrenals.

18-Hydroxylation of deoxycorticosterone was studies with rat or bovine adrenal mitochondria or with reconstituted systems obtained from these fractions. The reconstituted systems consisted of a partially purified preparation of cytochrome P-450 from rat adrenals and a partially purified NADPH-cytochrome P450 reductase preparation from bovine adrenals. In some experimenta a soluble cytochrome P-450 fraction from bovine adrenals was used. Adrenodoxine and adrenodoxine reductase were shown to be the active components of the NADPH-cytochrome P-450 reductase preparation. Optimal assay conditions were determined for 18-hydroxylation by the crude mitochondrial fraction as well as by the reconstituted systems. In the presence of excess NADPH-cytochrome P-450 reductase fraction, the rate of 18-hydroxylation was linear with time and with the amount of cytochrome P-450. In incubations with intact rat adrenal mitochondria to which Ca2+ and an excess NADPH had been added, NADPH-cytochrome P-450 reductase increased the rate of 18-hydroxylation about 100%, indicating that NADPH-cytochrome P-45o reductase was to some extent rate-limiting. The rate of 18-hydroxylation of deoxycorticosterone by the reconstituted system as well as by intact mitochondrial fraction was much higher than the rat of 18-hydroxylation of corticosterone and progesterone. When the cytochrome P-450 preparation from rat adrenals in the reconstituted system was substituted for cytochrome P-450 from bovine adrenals, the rate of 18-hydroxylation decreased considerably. Under all experimental conditions, the 18-hydroxylation of deoxycorticosterone occurred with a concomitant and efficient 11beta-hydroxylation. Provided the source of cytochrome P-450 was the same, the ratio between 11beta- and 18hydroxylation was constant under all conditions and was not significantly different in the presence of metopirone, carbon monoxide, cytochrome c or different steroids. It is suggested that identical or at least very similar types of cytochrome P-450 are involved in 11beta- and 18-hydroxylation of deoxycorticosterone.