Stimulation of cell growth and DNA synthesis by peripheral benzodiazepine

The effects of peripheral benzodiazepine receptor ligands on cell proliferation were evaluated. PK11195 increased the growth rate of C6 glioma cells by 20-30% in the nanomolar range in serum free medium. [3H]thymidine incorporation into C6 glioma cells also were increased 22% and 25% after treatment by PK11195 and Ro5-4864, respectively. The effect of PK11195 as a mitogenic agent was estimated by mitogenic agent was estimated by [3H]thymidine incorporation using Swiss 3T3 cells. PK11195 increased DNA synthesis 170% over control at 10 nM. Higher concentrations of benzodiazepines showed inhibition of the DNA synthesis. Peripheral benzodiazepine binding sites underwent downregulation after exposure to serum free medium or to 10 nM PK11195. These findings suggest that peripheral benzodiazepines may be involved in the regulation of cell proliferation as a growth factor in lower concentration and as a antiproliferative agent in higher concentration.     

Inhibition of embryonic cell aggregation by neoplastic cells.

The effects of normal and malignant cells on the aggregation of embryonic cells in gyratory shaker cultures were compared. The addition of 1 times 10-5 simian virus 40 (SV40)-transformed BALB/3T3 (SV40-3T3) cells to 6 times 10-6 embryonic neural retina cells caused a highly significant greater reduct on (22.7 percent) in aggregate diameter than the addition of untransformed BALB/3T3 (3T3) cells. The ratio of the number of single cells to the number of aggregates was significantly higher for cultures containing SV40-3T3 cells than for the cultures containing 3T3 cells. This effect was concentration dependent in the presence of cultured Ehrlich-Lettre hyperdiploid (ELD) ascites cells; however, media from ELD cell cultures or ELD cell sonicates resulted in aggregates of greater diameter and lower ratios of single cells to aggregates. This approach may provide a sensitive assay system for the interactions of tumor and other cells in vitro.     

Inhibition of DNA replication and growth of several human and murine neoplastic cells by aphidicolin without detectable effect upon synthesis of immunoglobulins and HLA antigens

Aphidicolin inhibits DNA replication and growth of all tested human and murine neoplastic cells including leukemic T- and B-lymphocytes and melanocarcinoma cells. The concentration of aphidicolin causing 50% inhibition of DNA synthesis in all of the tested neoplastic cell lines is similar to that necessary to inhibit DNA synthesis in HeLa cells by 50%. The mechanism of inhibition of DNA synthesis in neoplastic cells is again due to the inhibition of DNA polymerase alpha by aphidicolin. Aphidicolin at a concentration 100 times higher than that causing 50% inhibition of DNA synthesis and cell growth had no effect on total protein synthesis, on the secretion of immunoglobulins, or on the expression of HLA antigens which are involved in relevant phenomena of the immune response.     

Inhibition of DNA synthesis in rat hepatocytes by platelet-derived type beta transforming growth factor

Platelet-derived type beta transforming growth factor (TGF beta) is a potent inhibitor of DNA synthesis in primary monolayer cultures of adult rat hepatocytes. TGF beta induced a 50% inhibition of epidermal growth factor (EGF)-mediated DNA synthesis at approximately 5 X 10(12) M. This inhibition did not appear to be due to a delay in the peak of DNA synthesis or a toxic action, nor could it be overcome by increasing concentrations of the mitogens EGF, insulin, or glucagon. Inhibition was observed either when TGF beta and EGF were continuously present together in the culture medium or when TGF beta was added to the hepatocyte cultures after removal of the EGF stimulant. This observation together with a lack of an inhibitory effect of TGF beta on the binding of 125I-labeled EGF to hepatocytes in culture, suggests that the inhibitory action of TGF beta was not caused by a direct competition with EGF at the cell surface. TGF beta could not inhibit DNA synthesis once it had begun; however, the inhibitory action of TGF beta could be partially overcome by increasing amounts of conditioned medium produced by normal hepatocytes. Specific saturable receptors for TGF beta were found on the normal rat hepatocytes, but specific binding could not be detected on hepatocytes from regenerating liver. TGF beta is thus a potent inhibitor of EGF-induced DNA synthesis in adult rat hepatocytes. Its significance for growth control in vivo has yet to be assessed.     

Inhibition of growth of human small cell and non-small cell lung carcinomas by antagonists of growth hormone-releasing hormone (GH-RH)

Insulin-like growth factors-I and-II (IGF-I and IGF-II) may be involved in the proliferation of human lung carcinomas. The purpose of this study was to investigate the effects of two potent antagonists of growth hormone-releasing hormone (GH-RH), MZ-4-71 and MZ-5-156 on the growth of the H69 human small cell lung cancer (SCLC) and H157 non-SCLC (NSCLC) lines transplanted into nude mice or cultured in vitro. Nude mice bearing H69 and H157 tumors were treated for 3-5 weeks with MZ-4-71 or MZ-5-156 injected s.c. twice a day at a dose of 20 mu g/animal. Growth of H69 and H157 tumors in nude mice was significantly inhibited by MZ-4-71 and MZ-5-156 as shown by a reduction in tumor volume and weight. In animals bearing H157 NSCLC, treatment with MZ-4-71 decreased IGF-I and IGF-II levels in tumor tissue. Levels of IGF-I, but not of IGF-II in serum and liver tissue of H157 tumor-bearing nude mice treated with MZ-4-71 were decreased. High affinity binding sites for ICF-I were demonstrated on membranes of H69 and H157 tumors. In cell cultures, the proliferation rate of H69 SCLC cells was suppressed by 10(-7)-10(-5) M MZ-4-71, but H157 NSCLC line was only inhibited by 10(-5) M antagonist. Our findings demonstrate that the GHRH antagonists MZ-4-71 and MZ-5-156 can inhibit the growth of SCLC and NSCLC. This new approach to the management of lung cancer merits further investigation.     

The effect of vitamin A supplementation on serum retinol and retinol binding protein levels

A randomised double blind controlled trial was conducted to see whether vitamin A supplementation in the form of retinyl palmitate would increase the concentrations of serum retinol and retinol binding protein. A total of 376 people were studied and were allocated to one of 7 regimens covering doses of vitamin A from O (placebo) to 36,000 IU daily. Supplementation continued for 6 months and blood samples were collected immediately before the start of supplementation, after 3 months and after 6 months. There was a small but statistically significant increase in serum retinol levels associated with supplementation, but no significant increase in serum retinol binding protein. The extent of the increase in serum retinol was related to the extent of the supplementation. On average, for every 10,000 IU of retinyl palmitate per day, the serum retinol concentration increased by 13 micrograms/l after 3 months (an increase of 2%) and 12 micrograms/l after 6 months of supplementation (2% increase). All the regimens used showed no evidence of toxicity other than minor symptomatic and physical changes affecting the skin and mucous membranes.     

Inhibition of human lung cancer cell growth by angiotensin-(1-7)

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide hormone of the renin-angiotensin system with vasodilator and anti-proliferative properties. Human adenocarcinoma SK-LU-1 and A549 cells as well as non-small lung cancer SK-MES-1 cells were treated with serum in the presence and absence of Ang-(1-7), to determine whether Ang-(1-7) inhibits the growth of lung cancer cells. Ang-(1-7) caused a significant reduction in serum-stimulated growth in all three lung cancer cell lines. Treatment with Ang-(1-7) resulted in both a dose- and time-dependent reduction in serum-stimulated DNA synthesis in all three cell lines, with IC(50)'s in the sub-nanomolar range. The Ang-(1-7) receptor antagonist [D-Ala(7)]-Ang-(1-7) blocked the attenuation of the serum-stimulated DNA synthesis of SK-LU-1 cells by Ang-(1-7), while neither AT(1) nor AT(2) angiotensin receptor subtype antagonists prevented the response to the heptapeptide. MAS mRNA and protein, a receptor for Ang-(1-7), was detected in the three lung cancer cell lines, suggesting that the anti-proliferative effect of Ang-(1-7) in the cancer cells may be mediated by the non-AT(1), non-AT(2), AT((1-7)) receptor MAS. Other angiotensin peptides [Ang I, Ang II, Ang-(2-8), Ang-(3-8) and Ang-(3-7)] did not attenuate mitogen-stimulated DNA synthesis of SK-LU-1 cells, demonstrating that Ang-(1-7) selectively inhibits SK-LU-1 cancer cell growth. Pre-treatment of SK-LU-1 cells with 10 nM Ang-(1-7) reduced serum-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)1 and ERK2, indicating that the anti-proliferative effects may occur, at least in part, through inhibition of the ERK signal transduction pathway. The results of this study suggest that Ang-(1-7) inhibits lung cancer cell growth through the activation of an angiotensin peptide receptor and may represent a novel chemotherapeutic and chemopreventive treatment for lung cancer.     

Inhibition of human malignant neuroblastoma cell DNA synthesis by lipoxygenase metabolites of arachidonic acid

In vivo studies have shown that inhibitors of cyclooxygenase metabolism of arachidonic acid may diminish growth and metastasis of certain tumors. Because cyclooxygenase inhibition may increase the production of lipoxygenase products of arachidonic acid metabolism, we have investigated the effect of two such products, 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) on tumor cell proliferation in vitro. When neuroblastoma cells (SK-N-SH) in culture were treated with 12-HETE for 18 hr, incorporation of [3H]thymidine was inhibited up to 64% at concentrations from 20 to 50 microM. Under the same conditions, 15-HETE resulted in inhibition of up to 46%, while arachidonic acid had no apparent effect. When evaluated in the presence of serum, 12-HETE at a concentration of 120 microM produced a 20.6 +/- 2.8% (S.E.) inhibition of the increase in total DNA content over 48 hr, while 15-HETE at this concentration produced a 16.5 +/- 5.3% inhibition. We conclude that 12-HETE, the product of platelet lipoxygenase, and 15-HETE, a product of neutrophil and lymphocyte lipoxygenases, can inhibit human neuroblastoma cell growth in vitro and may play a role in the effect of cyclooxygenase inhibitors on tumor growth in vivo.     

Inhibition of human carcinoma cell growth and DNA synthesis by silibinin, an active constituent of milk thistle: comparison with silymarin

Several studies from our laboratory have shown the cancer chemopreventive and anti-carcinogenic effects of silymarin, a flavonoid antioxidant isolated from milk thistle, in long-term tumorigenesis models and in human prostate, breast and cervical carcinoma cells. Since silymarin is composed mainly of silibinin with small amounts of other stereoisomers of silibinin, in the present communication, studies were performed to assess whether the cancer preventive and anti-carcinogenic effects of silymarin are due to its major component silibinin. Treatment of different prostate, breast, and cervical human carcinoma cells with silibinin resulted in a highly significant inhibition of both cell growth and DNA synthesis in a time-dependent manner with large loss of cell viability only in case of cervical carcinoma cells. When compared with silymarin, these effects of silibinin were consistent and comparable in terms of cell growth and DNA synthesis inhibition, and loss of cell viability. Based on the comparable results of silibinin and silymarin, we suggest that the cancer chemopreventive and anti-carcinogenic effects of silymarin reported earlier are due to the main constituent silibinin.     

Inhibition of de novo pyrimidine nucleotide and DNA synthesis and growth of cultured Novikoff rat hepatoma cells and other cell lines by pyrazofurin (NSC 143095).

Pyrazofurin (PYF), a C-riboside, inhibited the replication of cultured Novikoff rat hepatoma cells, HeLa cells, and mouse L-cells at concentrations as low as 0.1 to 10 muM, but Novikoff cells were more sensitive than the cells of the other two cell lines. Inhibition of cell replication was completely prevented by the presence of 0.1 to 1 mM uridine in the medium, and partly by the presence of other pyrimidine, but not purine nucleosides. A 2- to 4-hr treatment of the cells with 10 muM PYF resulted in a 2-fold increase in the rate of incorporation of uridine into the acid-soluble pool and nucleic acids, while the rate of incorporation of adenosine into RNA was reduced about 85%. The incorporation of adenosine and deoxyuridine into DNA were reduced about 85 and 50%, respectively. The results are consistent with the view that PYF inhibits the de novo synthesis of pyrimidine nucleosides. The inhibition of cell replication seems to be due mainly to an inhibition of DNA rather than RNA synthesis, resulting from a rapid depletion of the pyrimidine deoxynucleotide pool, since addition of thymidine and deoxycytidine reversed the inhibition of DNA synthesis and cell replication by PYF. PYF must enter the cells to exert its toxicity since the toxicity of PYF was reduced 70 to 80% by the presence of 8 muM Persantin, a potent inhibitor of the facilitated and simple diffusion of various substrates, in the medium. If PYF is incorporated via normal nucleoside salvage pathways, its affinity for the nucleoside transport system(s) and kinases, must be low since, even at a concentration of 1 mM, it had only a slight effect on the initial rates of incorporation of various nucleosides into the nucleotide pool.     

Inhibition of the growth of transformed and neoplastic cells by the dipeptide carnosine.

Human diploid fibroblasts growth normally in medium containing physiological concentrations of the naturally occurring dipeptide carnosine (beta-alanyl-L-histidine). These concentrations are cytotoxic to transformed and neoplastic cells lines in modified Eagle medium (MEM), whereas these cells grow vigorously in Dulbecco''s modified Eagle medium (DMEM) containing carnosine. This difference is due to the presence of 1 mM sodium pyruvate in DMEM. Seven human cell lines and two rodent cell lines were tested and all are strongly inhibited by carnosine in the absence of pyruvate. Experiments with HeLa cells show that anserine is similar to carnosine, but D-carnosine and homocarnosine are without effect. Also, the non-essential amino acids alanine and glutamic acid contribute to the effect of pyruvate in preventing carnosine toxicity, and oxaloacetate and alpha-ketoglutarate can substitute for pyruvate. We have used mixtures of normal MRC-5 fibroblasts and HeLa cells to demonstrate that 20 mM carnosine can selectively eliminate the tumour cells. This has obvious implications which might be exploited in in vivo and in vitro studies. Carnosine is known to react strongly with aldehyde and keto groups of sugars by Amadori reaction, and we propose that it depletes certain glycolysis intermediates. It is well known that tumour cells are more dependent on glycolysis than normal cells. A reduction of glycolysis intermediates by carnosine may deplete their energy supply, but this effect is totally reversed by pyruvate.     

DNA synthesis enzymes and proliferating cell nuclear antigen in normal and neoplastic nerve cells.

The activity of nuclear DNA polymerases alpha, beta and delta/epsilon, uracil-DNA glycosylase, thymidine kinase and the presence of Proliferating Cell Nuclear Antigen (PCNA) have been examined in developing rat glial cells, in rat and human glioma, in human neuroblastoma and in differentiated neuroblastoma cell lines in vitro. During glial development the activity of all enzymes tested, except DNA polymerase beta, markedly decreased, suggesting their coordinate regulation in respect to the proliferative state of the cells. Glioma and neuroblastoma cell lines restore the enzymatic activities that were no longer expressed in normal adult cells. Neuroblastoma cell lines induced to differentiate in vitro by retinoic acid showed a decline of the activities of DNA polymerase alpha, DNA polymerase delta/epsilon, uracil-DNA glycosylase and thymidine kinase similar to that observed during in vivo differentiation. We also demonstrate that PCNA is not detectable in glial and neuronal cells at all developmental stages, but can be found in tumor nerve cells. A possible use of enzymatic assays or anti-PCNA antibodies to detect brain tumors is discussed.     

Inhibition of murine tumor growth and prostaglandin synthesis by indomethacin

Inhibition of the growth of a transplantable methylcholanthrene-induced tumor was achieved in mice by administering indomethacin or aspirin in the drinking water, these drugs having in common the ability to inhibit prostaglandin synthesis. Indomethacin was able to reduce the levels of prostaglandin E (PGE) in the tumor tissue. When tumors from these animals were cultured in vitro, the culture supernatant fluid from drug-treated animals showed lower levels of PGE released into the media, but after several days in culture, tumor cells from indomethacin-treated animals fully recovered their ability to produce PGE. The relative size of tumors in untreated animals was directly related to the amount of PGE activity present in these tumors.     

Prevention of dacarbazine damage of human neoplastic cell DNA by aphidicolin

Treatment of human neoplastic cells with dacarbazine both inhibits DNA synthesis and induces damage in the DNA. Lysis of cells in dilute alkali and subsequent electrophoretic analysis of the isolated DNA show that the DNA of treated cells includes a high molecular weight component and a population of 2-10-kilobase single-stranded DNA fragments while untreated cells contain only high molecular weight DNA. When DNA is pulse-labeled at the beginning of the dacarbazine treatment high amounts of small DNA fragments are seen but no labeled high molecular weight DNA. Moreover the DNA fragments are not formed in cells which are treated with aphidicolin before the addition of dacarbazine. Aphidicolin is a specific inhibitor of DNA polymerase alpha, the enzyme responsible for the replicative synthesis of DNA. We conclude that dacarbazine damages DNA only in cells which are synthesizing new DNA.     

Inhibition of breast cancer cell growth and induction of cell death by 1,1-bis(3'-indolyl)methane (DIM) and 5,5'-dibromoDIM

1,1-Bis(3'-indolyl)methane (DIM) and the 5,5'-dibromo ring substituted DIM (5,5'-diBrDIM) inhibited growth of MCF-7 and MDA-MB-231 breast cancer cells, and IC50 values were 10-20 and 1-5 microM, respectively, in both cell lines. DIM and 5,5'-diBrDIM did not induce p21 or p27 protein levels or alter expression of Sp1 or Sp3 proteins in either cell line. In contrast, 10 microM 5,5'-diBrDIM downregulated cyclin D1 protein in MCF-7 and MDA-MB-231 cells 12 and 24 h after treatment. DIM (20 microM) also decreased cyclin D1 in MCF-7 (24 h) and MDA-MB-231 (12 h), and the DIM/5,5'-diBrDIM-induced degradation of cyclin D1 was blocked by the proteasome inhibitor MG132. Both DIM and 5,5'-diBrDIM induced apoptosis in MCF-7 cells and this was accompanied by decreased Bcl-2, release of mitochondrial cytochrome c, and decreased mitochondrial membrane potential as determined by the red/green fluorescence of JC-1. DIM and 5,5'-diBrDIM induced extensive necrosis in MDA-MB-231 cells; however, this was accompanied by decreased mitochondrial membrane potential primarily in cells treated with 5,5'-diBrDIM but not DIM. Thus, DIM and 5,5'-diBrDIM induce cell death in MCF-7 and MDA-MB-231 cells by overlapping and different pathways, and the ring-substituted DIM represents a novel class of uncharged mitochondrial poisons that inhibit breast cancer cell and tumor growth.     

Inhibition of DNA and protein synthesis and cell division by photoactivated haematoporphyrin derivative in hamster ovary cells

Experiments were performed on cultured Chinese hamster ovary cells exposed to haematoporphyrin derivative (HpD) plus light, yielding survival rates of 40-100%. [3H]-thymidine, [3H]-tryptophan and [14C]-lysine incorporation were used to quantitate DNA and protein synthesis in surviving cells after exposure. Multiple experiments demonstrated 78% reduction in DNA synthesis during the first day after exposure to 20 micrograms ml-1 HpD plus 1140 Jm-2 light followed by progressive recovery to the normal rate after 4-6 days. Protein synthesis was somewhat less sensitive dropping by 54% initially and fully recovering by day 4. Although this cell line has a normal cycle time averaging approximately 15 h, cell division was rarely observed among lone surviving cells until 72 h after exposure. No inhibition was observed in cells exposed to HpD in the dark. These results indicate that photoactivated HpD has a wide spectrum of reversible nuclear and cytoplasmic effects even at sublethal doses. This is consistent with the notion that clinical photodynamic therapy is not likely to result in chronic morbidity.