2{alpha},3{alpha}-Epithio-5{alpha}-androstan-17{beta}-yl 1-Methoxycyclopentyl Ether in the Treatment of Advanced Breast Cancer --A Preliminary Clinical Trial--

A new orally active anti-estrogenic steroid, 2,3-epithio-5-androstan-17ß-yl1-methoxycyclopentyl ether (10364-S) was given to 41 advancedbreast cancer patients. Most patients were given a daily doseof 20 mg. The study was preliminary and not a controlled trialusing an already proven androgenic steroid. The remission rateon giving this compound to advanced breast cancer was 11/41or 26.8% and the average duration of the remission was 10.5months. Hoarseness (8/41, 19.5%) and hirsutism (5/41, 12.2%) were relativelyoften seen as virilizing side effects. No unfavorable effectson the hematopoietic organs, the liver or on calcium metabolismwere recognized in the study.     

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.     

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.     

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.     

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.     

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.     

Diagnostic Significance of Two Fetal Proteins, {alpha}f and r{beta}s, in Hepatoma and Other Neoplastic Diseases

Incidences of f and rßs components were studied inserum samples from patients with various malignant and non-malignantdiseases. With the micro-Ouchterlony method, af was detectedexclusively in cases of hepatocellular carcinoma of which theincidence was 83.5%, whereas the incidence of rßswas 47.7%. However, rßs was also detected in somemalignant disease other than hepatocellular carcinoma, withan incidence of 16.5%, and was also detected in a small numberof cases of non-neoplastic liver diseases. Though not tumor-specific, rßs as one of the fetalserum proteins can be detected in cases of non-f-producing hepatomaand in cases of malignancies other than hepatoma. Thus, thedetection of this protein may be of diagnostic significance.Possible mechanism of the appearance of this protein in theblood was discussed.     

UDP-GalNAc:Fuc{alpha}1-2Gal{alpha}1-3GalNAc transferase activity in hamster pancreatic cancers and in normal hamster alimentary tissues

Ductal adenocarcinomas induced by N-nitrosobis(2-oxopropyl)aminetreatment in Syrian hamsters produce blood group-A antigen,which is not present in normal hamster pancreas. To understandthe underlying mechanism of A antigen neoexpression in pancreaticcancer cells, we examined the activity of UDP-GalNAc: Fuc1–2Gal1–3GalNActransferase (A-transferase), the enzyme responsible for bloodgroup-A antigen production. The specific activity of A-transferasein the pancreatic cancers was 8 nmol/mg protein/h in membranepreparations, 0.3 nmol/mg protein/h in whole cell extracts,and undetectable in normal hamster pancreas. Significant A-transferaseactivity was found in normal tissues expressing blood group-Aantigen. Although both normal (gastric antrum, colon) and pancreaticcancer cells showed similar enzymatic characteristics (optimalpH, substrate affinity, optimal [Mn²⁺]), there was a differencein the requirement for divalent cations. The A-transferase incancer cells showed a more stringent requirement for Mn²⁺. Theseresults suggest that A-transferase is activated during nitrosamine-inducedpancreatic carcinogenesis, which results in the neoexpressionof blood group-A antigen. The difference in divalent cationrequirements between A-transferase activities of cancer andnormal cells may indicate that there are multiple A-transferasespresent in hamster tissues.     

17{beta}-Estradiol-induced cell transformation and aneuploidy of Syrian hamster embryo cells in culture

The ability of 17ß-estradiol to induce morphologicaltransformation of Syrian hamster embryo cells was examined anddose-dependent increases were observed over the concentrationrange of 1—10 µg/ml. However, treatment of the cellswith 17ß-estradiol failed to induce any detectableincreases in gene mutations, chromosome aberrations, sisterchromatid exchanges or unscheduled DNA synthesis. In contrast,over the dose range that was effective in inducing cell transformation,17ß-estradiol induced numerical chromosome changes(both chromosome gains and losses). These findings are similarto the reported observations with the synthetic estrogen, diethylstilbestrol,and support the hypothesis that aneuploidy induction is importantin cell transformation and possibly carcinogenesis induced byestrogens.     

X-ray crystallographic analysis of ({+/-})-7{beta},8{alpha}-dihydroxy-9{beta},10{beta}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene: molecular structure of a 'syn' diol epoxide

X-ray crystallographic analysis has been used to define themolecular structure of the cis (syn) diol epoxide, (±)-7ß,8-dihydroxy-9ß,10ß-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.The two hydroxyl groups are oriented equatorially to the tetrahydrobenzenering, contrary to predictions and there is no intramolecularhydrogen bonding in the structure.     

Treatment of metastatic renal cell carcinoma with constant-rate floxuridine infusion plus recombinant {alpha}2b-interferon

BACKGROUND:: Floxuridine (FUDR) and     

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.     

Cyr61 increases migration and MMP-13 expression via {alpha}v{beta}3 integrin, FAK, ERK and AP-1-dependent pathway in human chondrosarcoma cells

Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secretedand matrix-associated protein, which is involved in many cellularactivities such as growth and differentiation. However, theeffect of Cyr61 on migration activity in human chondrosarcomacells is mostly unknown. Here, we found that Cyr61 increasedthe migration and expression of matrix metalloproteinase (MMP)-13in human chondrosarcoma cells (JJ012 cells). RGD peptide, vβ3monoclonal antibody and mitogen-activated protein kinase (MEK)inhibitors (PD98059 and U0126) but not RAD peptide inhibitedthe Cyr61-induced increase of the migration and MMP-13 upregulationof chondrosarcoma cells. Cyr61 stimulation increased the phosphorylationof focal adhesion kinase (FAK) and extracellular signal-regulatedkinase (ERK). In addition, activator protein-1 (AP-1) decoyoligodeoxynucleotide also suppressed the MMP-13 messenger RNAand enzyme activity enhanced by Cyr61. Moreover, Cyr61 increasedthe binding of c-Fos and c-Jun to the AP-1 element on the MMP-13promoter. Taken together, our results indicated that Cyr61 enhancesthe migration of chondrosarcoma cells by increasing MMP-13 expressionthrough the vβ3 integrin receptor, FAK, ERK, c-Fos/c-Junand AP-1 signal transduction pathway. Abbreviations: AP-1, activator protein-1; AS, antisense; Cyr61, cysteine-rich 61; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; mAb, monoclonal antibody; MEK, MAPK kinase; MMP, matrix metalloproteinase; mRNA, messenger RNA; MS, missense; NF-B, nuclear factor-kappa B; ODN, oligonucleotide; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; qPCR, quantitative real-time polymerase chain reaction; siRNA, small-interfering RNA     

Human tumor cell strains both unable to repair O6-methylguanine and hypersensitive to killing by human {alpha} and {beta} interferons

Human brain tumor cell strains were previously found by othersto be sensitive to growth inhibition by human inter-feron-ß(HuIFN-ß). We noticed that the sensitive strains weresome that we had found deficient in the repair of O⁶-methyl-guanine(O⁶MeG), a characteristic of 20% of the human tumor cell strainswe have studied. We confirmed this sensitivity to HuIFN-ß,and have further shown that human brain tumor cells which repairO⁶MeG are resistant to the growth inhibitory effects of HuIFN-ß.In addition, treatment with HuIFN- or HuIFN-ß resultedin more killing (reproductive inactivation) of six human tumorcell strains deficient in repairing O⁶-methylguanine in DNAthan did such treatment of six strains of cells proficient insuch repair. Further, we found two human lines, altered to becomeO⁶MeG repair deficient after establishment of the primary tumorcell culture, that were resistant to interferon. IFN treatmentproduced no DNA damage detectable by either chemical or biologicalassays. It is suggested that the genes responsible for resistanceto IFN treatment and to agents that produce O⁶MeG are oftencoordinately shut down.