Chemopreventive effects of lupulone, a hop {beta}-acid, on human colon cancer-derived metastatic SW620 cells and in a rat model of colon carcinogenesis

The bitter acids of hops (Humulus lupulus L.) mainly consist of humulones or alpha-acids and lupulones or beta-acids. We aimed to evaluate the antiproliferative mechanisms of lupulones on a human metastatic colon carcinoma-derived cell line (SW620 cells) and to assess their chemopreventive effects in a model of colon carcinogenesis. SW620 cell growth was inhibited by 70% after a 48 h exposure to lupulones (40 microg/ml). Lupulones up-regulated the expression of Fas receptor (Fas) and Fas ligand (FasL) as well as TNF-related apoptosis inducing ligand (TRAIL)-R1 (DR4) and -R2 (DR5) receptor proteins, suggesting the involvement of Fas and TRAIL receptors-mediated pathways in lupulone-induced apoptosis. Lupulones also increased the mitochondrial membrane permeability. Colon carcinogenesis was initiated in Wistar rats by intra-peritoneal injections of azoxymethane (AOM), once a week for 2 weeks. One week after the last injection, rats received lupulones (0.001 or 0.005%) in drinking water, and AOM-control rats received the excipient. After 7 months of treatment, the colon of rats receiving 0.001 and 0.005% lupulones showed, respectively, a 30 and a 50% reduction (P < 0.05) of the number of preneoplastic lesions (aberrant crypt foci). In addition, we observed a drastic reduction (70-80%) of the total number of tumors in the colon of rats treated with lupulones when compared with the AOM control group. Lupulones induced apoptosis in SW620 colon-derived metastatic cells by activating both Fas and TRAIL death receptor signaling pathways, and antagonize at a low dose (4 mg/kg/day) colon cancer development. These observations suggest the use of lupulones for colon cancer chemoprevention trials.     

2{alpha} 3{alpha}-Epithio-5{alpha}-androstan-17{beta}-ol in Treatment of Gynecomastia

1. 1. The clinical effect of epitiostanol, a new anti-estrogenagent (2,3-epithio-5a-androstan-17ß-ol) against gynecomastiawas studied in comparison with dromostanolone propionate infifty-four patients ranging from twenty to fifty years in agewithout previous history of hormone therapy and with normalliver function. The experiment was performed for eight weeksby double blind methods in three dosage groups, epithiostanol10 mg, and 20 mg and dromostanolone propionate 50 mg.
2. 2. Epithiostanol20 mg was most effective with regards to effecton mass sizeand tenderness, (effective in 96%, 20/21), followedby 10 mgepitiostanol (effective in 89%, 16/18) and dromostanolonepropionate50 mg (effective in 89%, 16/18) in descending order.No sideeffects were observed in any of the three groups.
3. 3. Basedon the results of the present study, epitiostanol isconcludedto be at least as effective as dromostanolone propionateagainstgynecomastia and to be safe from the viewpoint of sideeffects.A satisfactory therapeutical effect on gynecomastiacan be expectedwith a weekly dosage of 20 mg of epitiostanolfor an administrationperiod of between five to eight weeks.

Present Address: Department of Surgery, Keio University Hospital,Shinanomachi, Shin-juku-ku, Tokyo, Japan.     

In situ analysis of transforming growth factor-{beta}s (TGF-{beta}1, TGF-{beta}2, TGF-{beta}3and TGF-3 type II receptor expression in malignant melanoma

We have analysed, by in situ hybridization, mRNA expressionof TGF-ß1, TGF-ß2, TGF-ß3, andof TGF-ß type II receptor in benign melanocytic naevi,primary melanomas, and in skin metastases of malignant melanomas.Our results show that melanoma progression correlates with overexpressionof TGF-ß. All skin metastases and most primary melanomasinvasive to Clark's level IV-V revealed specific TGF-ß2mRNA and protein expression. However, expression of this cytokinewas not observed in benign melanocytic lesions and was detectedonly in one of five early primary melanomas investigated. Someprimary melanomas and skin metastases also revealed specificTGF-ß1 mRNA signals although expression of this isoformwas not found in benign naevi. TGF-ß3 expression,which was only barely detectable in benign melanocytic lesions,was enhanced in some skin metastases. Interestingly, the epidermisoverlaying melanomas revealed lower levels of TGF-ß3mRNA expression than epidermis of healthy skin or epidermisadjacent to benign naevi, thereby suggesting that paracrinemechanisms between tumour cells and keratinocytes may influencemelanoma development. In primary melanomas TGF-ß typeII receptor mRNA signals were much more heterogeneously distributedwhen compared to benign melanocytic naevi, suggesting variabledegrees of TGF-ß resistance among melanoma cells withinindividual lesions. However, melanoma progression appeared notto be correlated with a complete loss of TGF-ß typeII receptor gene expression, since all skin metastases revealedclearly detectable although heterogeneous levels of TGF-ßtype II receptor mRNA expression.     

Enzymatic reduction of {beta}-ketonitrosamines

The reduction of N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-oxopropyl)propylamine(NOPPA) by hepatic and pancreatic cytosol and microsomes fromSyrian golden hamsters and Sprague-Dawley rats has been examined.All hepatic fractions reduced both substrates, although theactivity depended on the fraction tested and the cofactor employed(NADH or NADPH). Generally, hamster hepatic fractions containedhigher activity than the rat hepatic fractions and BOP was abetter substrate than NOPPA. Of the pancreatic fractions, onlycytosol exhibited reductase activity. The hamster cytosol wasable to utilise both cofactors, but the rat fraction exhibitedactivity only when NADPH was present. BOP was the better substratefor the pancreatic enzymes and in the presence of NADPH, therat and hamster activities were about equal. These results suggestthat the pancreatic reduction of BOP to HPOP is unlikely tobe a significant factor in the species-specific induction ofpancreatic cancer by BOP.     

Expression of cytokines, TNF-{alpha} and IL-1{alpha}, in MAM acetate and 1-hydroxyanthraquinone-induced colon carcinogenesis of rats

The expression of cytokines, TNF-     

Attenuation of proteasome-induced proteolysis in skeletal muscle by {beta}-hydroxy-{beta}-methylbutyrate in cancer-induced muscle loss

Loss of skeletal muscle is an important determinant of survival in patients with cancer-induced weight loss. The effect of the leucine metabolite beta-hydroxy-beta-methylbutyrate (HMB) on the reduction of body weight loss and protein degradation in the MAC16 model of cancer-induced weight loss has been compared with that of eicosapentaenoic acid (EPA), a recognized inhibitor of protein degradation. HMB was found to attenuate the development of weight loss at a dose greater than 0.125 g/kg accompanied by a small reduction in tumor growth rate. When EPA was used at a suboptimal dose level (0.6 g/kg) the combination with HMB seemed to enhance the anticachectic effect. Both treatments caused an increase in the wet weight of soleus muscle and a reduction in protein degradation, although there did not seem to be a synergistic effect of the combination. Proteasome activity, determined by the "chymotrypsin-like" enzyme activity, was attenuated by both HMB and EPA. Protein expression of the 20S alpha or beta subunits was reduced by at least 50%, as were the ATPase subunits MSS1 and p42 of the 19S proteasome regulatory subunit. This was accompanied by a reduction in the expression of E2(14k) ubiquitin-conjugating enzyme. The combination of EPA and HMB was at least as effective or more effective than either treatment alone. Attenuation of proteasome expression was reflected as a reduction in protein degradation in gastrocnemius muscle of cachectic mice treated with HMB. In addition, HMB produced a significant stimulation of protein synthesis in skeletal muscle. These results suggest that HMB preserves lean body mass and attenuates protein degradation through down-regulation of the increased expression of key regulatory components of the ubiquitin-proteasome proteolytic pathway, together with stimulation of protein synthesis.     

Engagement of I-branching {beta}-1, 6-N-acetylglucosaminyltransferase 2 in breast cancer metastasis and TGF-{beta} signaling

In this study, we have showed that GCNT2, a gene-encoding glucosaminyl (N-acetyl) transferase 2, I-branching enzyme, is overexpressed in highly metastatic breast cancer cell lines of human and mouse origin and basal-like breast tumor samples. GCNT2 expression is also significantly correlated to the metastatic phenotype in breast tumor samples. Functional studies showed that ectopic expression of GCNT2 enhances cell detachment, adhesion to endothelial cells, cell migration and invasion in vitro, and lung metastasis of breast cancer cells in vivo. Knockdown of GCNT2 expression decreases cell migration and invasion in vitro and lung metastasis in vivo. We have further shown the involvement of GCNT2 in the epithelial-to-mesenchymal transition (EMT). Specifically, the expression of E-cadherin is significantly changed upon GCNT2 expression at the protein level but not at the RNA level. Moreover, we have shown that GCNT2 is a direct target of the TGF-β-smad pathway and that change in GCNT2 expression modulates EMT induced by TGF-β1 treatment. Finally, we have shown that diminution of the glycosyltransferase activity of I-branching β-1, 6-N-acetylglucosaminyl transferase 2 (GCNT2) abrogates its cell migration and invasion-promoting function and synergistic effect with TGF-β to induce EMT. Our study for the first time showed that GCNT2 is a novel gene contributing to breast cancer metastasis with preferential expression in basal-like breast cancer. Moreover, we discovered that involvement of GCNT2 in EMT and TGF-β signaling, and further glycosylation modification of E-cadherin by GCNT2, are the underlying integrative mechanisms for breast cancer metastasis, implying that blocking TGF-β/GCNT2 signaling is a promising approach for targeting metastatic breast cancer.     

Comparative genotoxicity of 2,3'-O-cyclocytidine, {beta}-xylocytidine and 1-{beta}-D-arabinofuranosylcytosine in human tumor cell lines

We have Investigated the genotoxicity of two 3'-derivativesof cytidine, 2,3'-O-cyclocytidine (3'-cycloC) and ß-xylocytidine(xyloC), in human leukemia and solid tumor cell lines. Bothderivatives were found to be cytotoxic at micromolar concentrations.For example, in the alveolar tumor cell line A549 which wasincluded in all experiments as a reference, drug concentrationsrequired to induce 50% inhibition of cell growth (DM values)equalled 55 (iMfor 3'-cycloC and 80 µM for xyloC. Comparedwith the response of this reference cell line, none of the solidtumor cell lines tested—representing five different malignancies—displayedsignificant hypersensitivity to these drugs, while the acutelymphoblastic leukemia cell lines proved to be hypersensitive(range of D₅₀ values, 5–13 (µM). To gain insightinto the modes of cytotoxic action of xyloC and 3'-cycloC, wecompared the effect on DNA metabolism of these compounds withthat of 1-p-D-arabinofuranosylcytosine (araC), a potent inhibitorof semi-conservative DNA replication and long-patch excisionrepair. As seen with araC, the xylo compound strongly inhibitedboth DNA replicative synthesis and the repair of DNA damageinduced by UV light and ⁶⁰Co -radiation. In -irradiated A549cells, the extent of repair inhibition by 1 mM xyloC was 40%of that inhibited by araC, and concomitant exposure of the irradiatedcultures to xyloC plus araC gave rise to a synergistic response.Since araC was employed at a concentration (0.1 mM) which produceda maximal effect on DNA repair when applied alone, the observedsynergistic response implies that the mode of action of xyloCon DNA repair is different from that of araC. In contrast tothat observed with xyloC, 3'-cycloC proved to be a very weakinhibitor of DNA replication and repair, strongly suggestingthat the genotoxic action of the latter analog may be througha mechanism other than inhibition of DNA synthesis.     

CANCER BIOLOGY: Effects of dietary {beta}-carotene and selenium on initiation and promotion of pancreatic carcinogenesis in azaserine-treated rats

In the present study the effects of 0.1 or 1.0 g ß-carotene/kg diet (LßC or HßC) and 1.0 mg or 2.5 mgselenium/kg diet (LSel or HSel), as well as combinations ofthe respective low and high concentrations of ß-caroteneand selenium (LMix or HMix) on the initiation/early promotionphase or on the late promotion phase of pancreatic carcinogenesisin azaserine-treated rats, were investigated using cell proliferationand volumetric data of atypical acinar cell foci (AACF) as parameters.The present results indicate chemo-preventive effects of dietaryselenium, dietary ß-carotene and of their combinationon the development of acinar pancreatic lesions induced in ratsby azaserine. The inhibitory effect was most pronounced whenß-carotene and/or selenium were added to the dietsduring the late promotion phase of the carcinogenic process,although inhibition was also observed with these compounds whenthey were added to the diets during the first 5 weeks of thestudy only (initiation/early promotion phase). Neither in theinitiation/ early promotion phase nor in the late promotionphase was a dose-related trend observed. The multiplicitiesof AACF with a diameter over 1.0 mm and of carcinomas in situ(CIS), as well as the incidence of CIS were not significantlydifferent among the groups. However, in the late promotion experimenta dose-related decline in multiplicity could be observed inthe selenium supplemented groups and in the groups receivingcombinations of ß-carotene and selenium. Cell proliferationin azaserine-induced AACF, as estimated by the bromodeoxyuridine(BrdU) labeling index, was significantly higher in HßC,HSel, LMix and HMix groups (initiation/early promotion phase)as well as in HßC, LSel, HSel, LMix and HMix groupsGate promotion phase) than in high fat controls. From the presentresults it can be concluded that: (i) ß-carotene andselenium have inhibitory effects on pancreatic carcinogenesisinduced in rats by azaserine; (ii) the most clear effects wereobserved when selenium was given as such, or in combinationwith ß-carotene during the late promotion phase; and(iii) ß-carotene and selenium stimulate cell proliferationin AACF.     

Autocrine production of TGF-{alpha} and TGF-{beta} during tumour progression of rat oral keratinocytes

This study describes a new technique to separate transforminggrowth factor- (TGF-) and transforming growth factor-ß(TGF-ß) from culture supernatants using ion exchangechromatography; assays of competitive inhibition of ligand bindingwere used to quantify the amount of growth factor. The methodwas simple, inexpensive and did not require large volumes ofculture medium. The autocrine production of TGF- and TGF-ßwas examined in oral keratinocyte cell lines derived from thepalatal and lingual mucosa of rats painted with the carcinogen4-nitroquinoline N-oxide (4NQO). Escape from cellular senescence(immortality) was associated with a marked increase in TGF-production (cell line R2P) but tumour progression, as reflectedby the development of anchorage independence in agarose gelsand tumorigenicity in athymic mice, did not result in a consistentincrease or decrease of TGF- production compared to normals.Four cell lines(R8AP, R1T, R3T, R1P), with different functionalcellular phenotypes, produced two to three times more TGF- thannormals. TGF- production was inversely correlated to epidermalgrowth factor cell surface receptor expression. The autocrineproduction of TGF-ß was variable with the majorityof cell lines producing markedly little TGF-ß threecell lines (R4T, R8BP, R9T) produced more TGF-ß thannormals. The production of TGF-ß was unrelated totumour progression, the expression of TGF-ß cell surfacereceptors or TGF- production. The results indicate that theautocrine production of TGF- and TGF-ß are not accuratemarkers of tumour progression in the rat 4NQO model of oralcarcinogenesis.     

Induction of colon mucosal β-glucuronidase production as a mechanism for 1,2-dimethylhydrazine colon carcinogenesis

Although early studies in germ-free rats showed almost complete dependence on dimethylhydrazine (DMH) colon carcinogenesis upon the presence of colon bacteria, no adequate explanation was given for the 20% tumor incidence observed in germ-free animals. Bacterial activation of liver microsomal products releasing active proximate carcinogens has been the accepted reason for the exquisite specificity DMH has for the colon. Recent work, including the present study, show the colon mucosa is capable of metabolizing carcinogens and activating conjugating forms metabolized in the liver independent of the intestinal microflora. Mucosal β-glucuronidase production was assayed in coded, scraped mucosa samples from the duodenum/jejunum, ileum, right colon, and left colon of normal and DMH-treated rats. Normal mucosal β-glucuronidase production was highest in the left colon followed by the right colon, duodenum, and ileum, respectively. Enzyme production in the left colon was significantly increased 24 hours after injection of 25 mg/kg body weight DMH. No elevation was seen in other mucosal samples. Metabolism of DMH to oxidated forms conjugated to glucuronic acid is well established. Thus, this study offers a possible role for carcinogen, induction of a metabolic enzyme in its target tissue.     

The reaction of {beta}-propiolactone with derivatives of adenine and with DNA

The reaction of deoxyadenosine with ß-propiolactoneproduces two derivatives. One is l-(2-carboxyetbyl)-2'-deoxyadenosine(CEdA) first described by Maté, et al. The proposed structurefor the other is 3-(ß-D-2-deoxy-ribosyl)-7, 8-dihydropyrimido-[2,l-i]purine-9-one (dDPP). Spectral characteristics of both compoundsare presented. These include u.v. spectra of each in acidic,neutral and alkaline solutions, i.r. spectra, fluorescence spectra,and n.m.r. spectra. The extinction coefficient for CEdA is 12,900 M^-1cm^-1 at 258 nm and that for dDPP is 12, 400 M ^-1cm^-1at 305 nm. The dDPP can be converted to CEdA by mild acid hydrolysis,and the CEdA can be converted to dDPP by reaction with a car-bodiimidederivative. When poly A was reacted with ß-propiolactone,the yield of dDPP in the polymer was 7–9%. When double-strandedDNA was alkylated by [³H]ß-proplolactone at relativelyhigh concentrations and then acid hydrolyzed to separate 1-(2-carboxy-ethyl)adenine(CEA) and 7-(2-carboxyethyl)guanine (CEG), a CEA to CEG ratioof up to 0.62 was obtained. With relatively low concentrationsof [³H]propiolactone, the yield of CEA was low with double-strandedDNA but was 5–6 fold greater with single-stranded DNA.     

Suppression of mitogenic activity by stable expression of the regulatory domain of PKC{beta}

The amino-terminal regulatory domain portion of each proteinklnase C (PKC) family member (which in the case of PKCß1includes the pseudosubstrate, C1, VI and C2 domains) plays animportant role in regulating the kinase activity of the carboxyI-terminalcatalytic domain. To examine the possibility that this regulatorydomain region (designated ‘PAT’) might have biologicalfunctions independent of the catalytic domain, we have developedderivatives of R6 cells which stably express a truncated PKCß1cDNA that encodes the amino-terminal 317 amino acids, includingthe entire regulatory domain. These R6-plPAT cells express abundantamounts of a 38 kDa protein which binds a labeled phorbol ester,but lacks protein kinase activity. In contrast to the 79 kDaPKCpi holoenzyme which, when overexpressed in R6 cells, is foundmostly in the cytosol, the 38 kDa PAT protein is predominantlyassociated with the particulate subcellular fraction. Furthermore,the PAT protein fails to show down-regulation following treatmentof R6-plPAT cells with 12-O-tetradecanoylphorbol-13-acetate(TPA). Evidence is also presented that TPA-stimulated growthis suppressed in R6-plPAT cells. These findings suggest thatthe PKCß1 regulatory domain could be involved in thesuppression of mitogenic signaling.     

Overexpression of protein kinase C {beta}1 in the SW480 colon cancer cell line growth suppression

Using a retroviral vector system we have established derivatiesof the E8 subclone of the human colon cancer cell line SW480that stably overproduce a full-length rat cDNA ecncoding theß1 isoform of protein kinase C(PKCß1). Incontract to vectrol control cells, when treated with the tumorpromoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), the overexpressingcell lines displayed a striking increase in doubling time, anda decrease in saturation density. Western blot analysis indicatedthat treatment with TPA was also associated with translocationand partial downregulation of the exogenous PKCß1in the over-ex-pressor cell lines. These results extend previousevidence that PKCß1 can inhibit the growth of theHT29 human colon cancer cell line. The HT29 cells have a normalc-k-ras oncogene but the SW480 cells used in the present studyhave an activating mutation in this oncogene. Thus PKCß1can function as a suppressor in both types of colon cancer cells.     

Inhibition of 7,12-dimethylbenzanthracene-induced rat mammary tumorigenesis by 2,5-di-O-acetyl-D-glucaro-1, 4:6,3-dilactone, an in vivo {beta}-glucuronidase inhibitor

2,5-Di-O-acetyl-D-glucaro-1,4:6,3-dilactone (DAGDL) is a slowrelease form of D-glucaro-1,4-lactone (GL), a non-toxic naturalinhibitor of ß-glucuronidase. When administered orallyto female rats in conjunction with a carcinogenic dose of 7,12-dimethylbenzanthracene(DMBA), this compound caused a 70% reduction in the number ofrats with mammary tumors and 72% reduction in the number ofmammary tumors per rat. Co-administration also reduces the inductionby DMBA of a 60 kd oncofetal protein, previously shown to beassociated with carcinogenesis and tumorigenesis. DAGDL administrationdepressed ß-glucuronidase activity both in the absenceand presence of concurrent treatment with DMBA and also markedlyreduced binding of DMBA to organ DNA. The anti-carcinogeniceffect of DAGDL appears to be independent of route of administrationof DMBA. It is proposed that inhibition of ß-glucuronidaseincreases the proportion of DMBA which is sequestered and excretedas the glucuronide and therefore unavailable for activationto the proximal carcinogen.     

Effect of exogenous {beta}-glucuronidase on the carcinogenicity of 1,2-dimethylhydrazine in rats: evidence that carcinogenic intermediates form conjugates and act through their subsequent enzymatic release

Albino, outred 3-month-old rats were given a single s.c. doseof 1,2-dimethyihydrazine dihydrochioride (DMH; 100 mg/kg) and,6 or 24 h later, an i.v. dose of bovine liver ß-glucuronldase(3 x 10⁴ Fishman units). After this treatment, the incidenceof tumours of the large intestine and Zymbal gland, and of cystocholangiomaswas similar to that found in rats treated with DMH alone; theincidence of malignancies in various other tissues was considerablyhigher than that in rats treated only with DMH, especially inanimals exposed to ß-glucuron1dase 24 h after administrationof DMH. ß-Glucuronidase itself had no carcino genicactivity. The broadening of the spectrum of malignant tumoursproduced in DMH-treated rats by administration of ß-glucuronidaseindicates that the carcinogenic effect of DMH may be exertedthrough formation of comparatively stable conjugates of itsmetabolites and their enzymic release in target tissues. Theapproach used in this study could be helpful in investigatingthe formation of conjugates from other carcinogens.     

Effect of {beta}-carotene on cell cycle progression of human fibroblasts

The uptake of ß-carotene (BC) and its effect on thecell cycle progression of normal human fibroblasts in primaryculture were investigated by using two different delivery methods:exposure to BC solubilized in the organic solvent tetrahydrofuran(THF) or to BC incorporated into dipalmitoylphosphatidylcholine(DPPC) liposomes. Cell cycle progression was evaluated by immunofluorescencedetection and flow cytometric analysis of the proliferatingcell nuclear antigen (PCNA). In contrast to THF, which induceda marked reduction in the number of cells in S phase and inthe extent of PCNA immunolabeling, DPPC liposomes proved tobe an effective delivery system that does not interfere withcell proliferation. Cellular uptake of 0.23 nmol/10⁶ cells wasfound after 24 h incubation in BC-containing DPPC liposomes.This value increased to 1.2 nmol/10⁶ cells after 72 h. Afterthe first day of incubation, the number of cells in S phasewas reduced by 50%, with a consequent accumulation of cellsin G₁ phase. This effect was maintained up to 3 days incubation,with no detectable effects on cell viability. This cell cycledelay was found to be reversible, returning the percentage ofcells in S phase to the control value 24 h after removal ofBC from the medium. In order to determine whether the activityof BC could be attributed to the molecule itself or to its conversioninto retinoids, the production of BC metabolites was assessed.Analysis of cellular levels of retinoids failed to demonstratethe presence of retinal, retinol, retinoic acid or retinyl estersduring an incubation period of 6 days. These results suggestthat in normal human fibroblasts, BC induces a cell cycle delayin the G₁ phase and that this effect is independent of conversionto known retinoids.