Nai-Sum Wong

Adiponectin Haploinsufficiency Promotes Mammary Tumor Development in MMTV-PyVT Mice by Modulation of Phosphatase and Tensin Homolog Activities

Background Adiponectin is an adipokine possessing beneficial effects on obesity-related medical complications. A negative association of adiponectin levels with breast cancer development has been demonstrated. However, the precise role of adiponectin deficiency in mammary carcinogenesis remains elusive. Methodology/Principal Findings In the present study, MMTV-polyomavirus middle T antigen (MMTV-PyVT) transgenic mice with reduced adiponectin expressions were established and the stromal effects of adiponectin haploinsufficiency on mammary tumor development evaluated. In mice from both FVB/N and C57BL/6J backgrounds, insufficient adiponectin production promoted mammary tumor onset and development. A distinctive basal-like subtype of tumors, with a more aggressive phenotype, was derived from adiponectin haplodeficient MMTV-PyVT mice. Comparing with those from control MMTV-PyVT mice, the isolated mammary tumor cells showed enhanced tumor progression in re-implanted nude mice, accelerated proliferation in primary cultures, and hyperactivated phosphatidylinositol-3-kinase (PI3K)/Akt/beta-catenin signaling, which at least partly attributed to the decreased phosphatase and tensin homolog (PTEN) activities. Further analysis revealed that PTEN was inactivated by a redox-regulated mechanism. Increased association of PTEN-thioredoxin complexes was detected in tumors derived from mice with reduced adiponectin levels. The activities of thioredoxin (Trx1) and thioredoxin reductase (TrxR1) were significantly elevated, whereas treatment with either curcumin, an irreversible inhibitor of TrxR1, or adiponectin largely attenuated their activities and resulted in the re-activation of PTEN in these tumor cells. Moreover, adiponectin could inhibit TrxR1 promoter-mediated transcription and restore the mRNA expressions of TrxR1. Conclusion Adiponectin haploinsufficiency facilitated mammary tumorigenesis by down-regulation of PTEN activity and activation of PI3K/Akt signalling pathway through a mechanism involving Trx1/TrxR1 redox regulations.

Phosphatidylinositol 3-kinase and protein kinase C are required for the inhibition of caspase activity by epidermal growth factor

The mechanism by which growth factors exert an anti‐apoptotic function on many cell types is not well understood. This issue is addressed in relation to epidermal growth factor (EGF) which inhibits apoptosis induced by staurosporine or wortmannin in an epithelial tumour cell line (CNE‐2). The presence of EGF substantially reduced the in vitro Ac‐DEVD‐AMC hydrolytic activity and almost completely suppressed the intracellular cleavage of poly(ADP‐ribose) polymerase in staurosporine‐ or wortmannin‐treated cells. Staurosporine but not wortmannin caused the intracellular proteolytic processing of pro‐caspase‐3 and this event was transiently inhibited by EGF. Staurosporine‐induced apoptosis was not inhibited by EGF in the presence of wortmannin or LY294002. Similarly, EGF failed to inhibit wortmannin‐induced apoptosis in the presence of staurosporine, chelerythrine chloride or Gö6850. These results suggest that phosphatidylinositol 3‐kinase and protein kinase C play a role in the survival function of EGF but the reduction of cellular caspase activity cannot be satisfactorily explained by a lack of pro‐caspase‐3 activation.

The PERK/eIF2α signaling pathway of Unfolded Protein Response is essential for N-(4-hydroxyphenyl)retinamide (4HPR)-induced cytotoxicity in cancer cells

N-(4-hydroxyphenyl)retinamide (4HPR) is a synthetic retinoid that has been tested in clinical trials as a cancer chemopreventive drug. 4HPR is cytotoxic to cancer cells but the underlying molecular mechanisms are at present only partially understood. Here we demonstrate that in the human cervical cancer cell line HeLa and the human leukemia cell line HL-60, 4HPR caused rapid, Reactive Oxygen Species (ROS)-dependent activation of the Unfolded Protein Response (UPR). In HeLa cells, 4HPR was shown to induce cell death and activation of procaspases. These effects of 4HPR could be abolished by the over-expression of dominant negative mutants of PERK or eIF2 alpha. HeLa cells incubated with 4HPR were found to form autophagosomes that were also mediated by the PERK/eIF2 alpha pathway. While 4HPR-induced cell death could be significantly prevented by the presence of specific caspase inhibitors, 3-methyladenine (3-MA) that inhibits autophagosome formation enhanced 4HPR-induced cell death. Examination of individual 4HPR-treated HeLa cells revealed that those without the development of autophagosomes hence exhibiting an incomplete UPR were caspase-active and were not viable, while those with autophagosomes were caspase-inactive and retained cell viability. Our data suggest that the PERK/eIF2 alpha pathway is essential for the cytotoxicity of 4HPR that targets on cancer cells with malfunctional UPR.

Upregulation of GADD153 expression in the apoptotic signaling of N‐(4‐hydroxyphenyl)retinamide (4HPR)

The molecular basis for the pharmacologic effects of N‐(4‐hydroxyphenyl)retinamide (4HPR) was investigated by studying the gene(s) that this compound may upregulate in cultured human epithelial tumor cells. Treatment of the cultured human nasopharyngeal carcinoma‐derived cells (CNE3) with 4HPR caused modest cell‐cycle arrest at G1 and apoptosis. The mRNA levels of a total of 20 genes were downregulated with the majority of them involved in cell cycle‐related functions. Only the mRNA level of the growth arrest and DNA‐damage inducible gene (gadd153) was upregulated by approximately 7‐fold, with a concomitant increase in intracellular protein level. Similar upregulation of gadd153 by 4HPR was observed in HeLa and 2 other tumor cell lines. The 4HPR‐induced apoptosis was markedly enhanced in the CNE3 cells that transiently overexpressed the gadd153 protein. Unlike 4HPR, all‐trans‐retinoic acid (ATRA) had no effect on the mRNA or protein level of gadd153. The ability of 4HPR and ATRA to stimulate the promoter activity of gadd153 was then examined. In the HeLa cells, both 4HPR and ATRA caused a 2‐ to 4‐fold stimulation of the promoter activity of gadd153, but similar to the CNE3 cells, ATRA was incapable of upregulating the protein level of gadd153. This is the first demonstration that gadd153 is a 4HPR‐responsive gene in tumor cells and may have a functional role to play in 4HPR‐induced apoptosis. Furthermore, our data suggest that the expression of gadd153 can be regulated by 4HPR at the transcriptional level.

Lithium attenuates the effects of dynorphin A(1–13) on inositol 1,4,5-trisphosphate and intracellular CA2+ in rat ventricular myocytes

When rat ventricular myocytes were stimulated with dynorphin A(1-13), a transient and rapid increase followed by a sustained and prolonged elevation in the intracellular levels of inositol 1,4,5-trisphosphate ¿Ins(1,4,5)P3¿ was observed. The responses were dose-related and abolished by nor-binaltorphimine. In the presence of lithium and absence of extracellular free inositol, the initial rapid elevation in Ins(1,4,5)P3 remained the same, but the second phase of sustained and prolonged elevation was abolished. Under this condition, the elevation in cytosolic free Ca2+ ([Ca2+]i) was reduced significantly although there was still a detectable elevation over a time period when the Ins(1,4,5)P3 was at the basal level. The responses in Ins(1,4,5)P3 and [Ca2+]i were not affected by lithium when stimulation of ventricular myocytes with dynorphin A(1-13) was performed in the presence of extracellular inositol. The data suggest that in rat ventricular myocytes, the kappa-opioid receptor agonist stimulated mobilization of [Ca2+]i was mediated mainly by Ins(1,4,5)P3.

Epidermal growth factor induces Gadd45 (growth arrest and DNA damage inducible protein) expression in A431 cells

Epidermal growth factor (EGF) receptor over-expressing, p53-deficient A431 cells response to toxic dose of EGF by G1 arrest and apoptosis. Applying cDNA expression array technology we demonstrated that EGF increased the levels of Gadd45 mRNA. Northern blot and Western blot analyses confirmed that both Gadd45 mRNA and protein were increased. Concurrently half-lives of Gadd45 mRNA and protein also increased. Nuclear run-on experiments did not show a large increase of Gadd45 mRNA transcription rate. Gadd45 mRNA and protein started to increase after 1 h of EGF treatment and remained high for up to 10 h. We have also confirmed previous studies which showed that in EGF-stimulated A431 cells p21(Cip1/Waf1) (cyclin-dependent kinase interacting protein 1) was up-regulated within the same time frame. Thus it appears that in addition to inducing G2 arrest by directly disrupting Cdc2/Cyclin B1 complex in genotoxic-stressed cells, Gadd45 may also participate in G1 arrest in growth factor overexposed cells.

Signal transduction in the cardiac k-receptor.

The presence of k-binding sites in the heart suggests a regulatory role of k-receptor in the cardiac functions. Recent studies have provided evidence that activation of cardiac k-receptor elevates intracellular free calcium ([Ca2+]i) by increasing mobilization of calcium from the intracellular store. The mobilization of intracellular calcium results from an increased production of inositol-1,4,5-trisphosphate (IP3). The increase in [Ca2+]i may manifest in cardiac arrhythmias while the depletion of calcium from the intracellular store may reduce the contractility elicited upon depolarization. The responses of IP3/[Ca2+]i are significantly attenuated after development of tolerance to k-opioids due mainly to the impairment of postreceptor events. The attenuated responses of IP3/[Ca2+]i to k-receptor activation may be responsible for the failure of the k-agonists to induce cardiac arrhythmias and to reduce electrically induced calcium transients in the ventricular myocytes.

Identification of plasmin as the major contaminant in immunoglobulin preparations

A proteolytic enzyme could be isolated from rabbit serum by means of DEAE cellulose, Protein A-bound Sepharose and lysine-bound Sepharose chromatographies. This enzyme was found to be the major protease contaminating IgG preparations of rabbit serum. This enzyme was identified as plasmin because it displayed an apparent Mr of 90,000 on nonreduced SDS polyacrylamide gel electrophoresis, was able to directly lyse fibrin and the chromogenic substrate H-D-Val-Leu-Lys-p-nitroanilide, and was stable after heating at 56 degrees for 30 min but broke down at 80 degrees. Its Km toward the chromogenic substrate was 0.35 mM, which agreed well with the published value for plasmin.

Demonstration of the presence of plasminogen activator in human small intestine

A cytosolic fraction of human small intestine was prepared. It contained esterase activity toward N-alpha-acetyl-lysine-methyl ester and amidolytic activities toward substrates S-2238, S-2288 and S-2251. In addition there was present a plasminogen activator activity which could cleave plasminogen to produce plasmin and the plasmin hydrolysed the same chromogenic substrates. Plasmin generation was also followed by a time-dependent hydrolysis of 125-I labeled plasminogen or monitored by fibrin-agar plate. The plasminogen activator was related to urinary urokinase immunologically. Anti-urokinase IgG cross-reacted with cytosolic fraction in double immunodiffusion. When the cytosolic fraction was electrophoresed in discontinuous polyacrylamide gel, two regions of hydrolytic activity toward the urokinase-specific substrate S-2444 were found. The activity of one of these regions could be completely inhibited by anti-urokinase while the other was not. The plasminogen activator was partially purified by ammonium sulfate precipitation and Concanavalin A-bound Sepharose chromatography.