Down-regulation of transforming growth factor-β and interleukin-10 secretion from malignant glioma cells by cytokines and anticancer drugs

The effect of treatment with interleukin-1 (IL-1), interferon- (IFN-), vincristine, and etoposide was evaluated on the secretion of transforming growth factor- (TGF-) and IL-10 and the expression of major histocompatibility complex (MHC) class I, intercellular adhesion molecule-1 (ICAM-1), and CD80 molecules by malignant glioma cells. Five malignant glioma cell lines were treated with IL-1, IFN-, and/or anticancer agents (vincristine and etoposide). Combined treatment with IL-1 and IFN- caused greater inhibition of TGF- secretion compared to treatment with IFN-, and almost the same levels of inhibition as treatment with vincristine and etoposide. The greatest inhibition of TGF- secretion was achieved by treatment with all agents. Low levels of IL-10 secretion were determined in two out of five malignant glioma cell lines. This IL-10 secretion was inhibited by treatment with IL-1, IFN-, vincristine, and/or etoposide. Treatment with both cytokines and anticancer agents increased the expression of MHC class I and ICAM-1 in all tumor cell lines. The mean increase of expression of MHC class I was 50% and that of ICAM-1 was 12-fold. No tumor cell lines expressed CD80 molecules on the cell surface, and no treatment caused CD80 expression. These results suggest that TGF- and IL-10 secretion by malignant glioma cells can be suppressed by treatment with a combination of IL-1, IFN-, vincristine, and etoposide, and the treatment up-regulates MHC class I and ICAM-1 expression on tumor cells. These results have implications for immunotherapy and chemotherapy in patients with malignant tumors.     

Interaction of transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) in human glioma cells

Gliomas are characterized by a deregulation of growth factor production and growth factor receptors expression, e.g. overproduction of the cytokine transforming growth factor- (TGF-) and overexpression/constitutive activation of receptors for the epidermal growth factor (EGF). Potential interactions of such growth factors and their signaling cascades could enhance the malignancy of these tumors. Therefore, we investigated the effects of TGF- and EGF alone and in combination on the proliferation of glioma cells cultivated from eight solid human WHO grade IV gliomas and one glioma cell line, analyzed the expression and intactness of the TGF--signaling molecules Samd-4 and -2, and the phosphorylation of the EGF-signaling kinases ERK 1/2. The effects were divergent and complex: Whereas EGF mostly stimulated glioma cell proliferation, TGF- either enhanced, inhibited or had no significant effect on proliferation. In combination, co-stimulation and inhibition of the EGF-induced mitogenic activity could be observed. Smad-4/-2 were expressed in all glioma cells, one point mutation at base 1595 in Smad-4 did not affect its protein sequence. In part of the glioma cells, reduced phosphorylation of ERK 1/2 and expression of cyclin-dependent kinase inhibitor 1 or p21 was observed in co-stimulation experiments. These experiments show that TGF- can inhibit EGF-mediated effects only in some gliomas, whereas it enhances it in others. The interaction of both factors is very complex and varies between different gliomas.     

Effect of irradiation on transforming growth factor-β secretion by malignant glioma cells

Glioblastoma cells secrete transforming growth factor- (TGF-), whichhas a variety of immunosuppressive properties. We investigatedthe effect of irradiation TGF- secretion by malignantglioma cells. Three malignant glioma cell lines (T98G,A172, KG-1-C) were cultured and irradiated using 10and 50 Gy Linac radiation. After further culturefor 36 hours in serum-free culture medium, thesupernatants were collected. The TGF- activity in theculture supernatants was determined using a specific bioassay.The levels of the active form and totalTGF- in the supernatants from irradiated malignant gliomacells decreased compared to those from un-irradiated cells.However, since irradiation inhibited the growth of tumorcells, the amount of TGF- secretion per cellin irradiated cells tended to increase after irradiation.These results suggest that malignant glioma cells canstill secrete TGF- and activate latent TGF- evenafter large dose irradiation, despite the inhibition oftumor growth.     

Effects of Thalidomide on Parameters Involved in Angiogenesis: An in vitro Study

In an attempt to elucidate the mechanism(s) of action of thalidomide, a reportedly antiangiogenic molecule recently tested in the treatment of relapsing malignant gliomas, we performed an in vitro study on the following parameters: (a) effect of thalidomide on proliferation of endothelial cells; (b) effect of thalidomide on expression of v3 integrin on the surface of endothelial cells; (c) effect of thalidomide on the release by endothelial cells of MMP-2, IL-8 and TNF-. The results show that thalidomide inhibits endotelial cell proliferation induced by bFGF and VEGF, more so if the cells are grown on vitronectin; moreover, treatment with thalidomide reduces the release of MMP-2 and IL-8 by endothelial cells, suggesting a further pathway for the antiangiogenic activity of drug. On the other hand, thalidomide does not modify expression of v3 on endothelial cells.     

Transforming growth factor beta and the cell surface in tumor progression

Summary Type 1 transforming growth beta (TGF-1) is a multifunctional regulator of cellular differentiation, motility and growth. It is capable of inhibiting or stimulating these processes depending on cell type, cell density, culture conditions and TGF-1 concentration. TGF-1 regulates growth, in part, by inducing the expression and secretion of various types of collagen, which participate in the control of cell adhesion and migration, as well as growth. TGF-1 also regulates cell growth by controlling the response to epidermal growth factor (EGF) and other growth factors, in ways that can either decrease or increase their growth-promoting effects. Alterations in both negative and positive growth responses to TGF-1 play important roles in tumor progression. Loss of sensitivity to growth inhibition by TGF-1 can occur as a result of decreased expression of collagen. Acquisition of sensitivity to growth stimulation, and autocrine transformation by TGF-1, are associated with aberrant EGF receptor regulation. Aberrant growth factor receptor regulation by TGF-1 may be mediated by a protein kinase C (PKC)-dependent pathway which inhibits degradation of growth factor receptor/ligand complexes. The evidence reviewed is consistent with a minimal two-step mechanism for autocrine transformation, which involves production of growth factor and enhanced cellular response as a result of aberrant membrane traffic. Defects in membrane traffic regulation may provide an explanation for common alterations in tumor cell response to both multiple growth inhibitors and growth stimulators, and may also suggest novel approaches to cancer chemotherapy.     

Sex hormone-induced mammary carcinogenesis in female Noble rats: Expression of TGF-β1 and its receptors, TGF-α, and EGF-R in mammary carcinogenesis

We have established a Noble rat model to explore the mechanisms of hormonal mammary carcinogenesis, in which the role of androgen in promoting mammary carcinogenesis was highlighted. We have also established that stromal–epithelial interactions may be responsible for the promotional effects of testosterone in mammary carcinogenesis. Based on these understandings, in the present study we examined the expression of transforming growth factor beta-1 (TGF-₁) and its receptors (TGF- RI, TGF- RII), transforming growth factor alpha (TGF-), and epidermal growth factor receptor (EGF-R) in 'pre-malignant' mammary glands treated with different protocols of sex hormones, as well as in mammary cancers. We observed that TGF-₁ was strongly expressed in most mammary tumors, whereas TGF- RI and TGF- RII were negative in most mammary tumor cells. The results from comparative study of 'pre-malignant' glands further showed that when the animals were treated with testosterone, either alone or in combination with 17-estradiol, the mammary gland epithelial cells expressed high levels of TGF-₁. This over-expression of TGF-₁ can be blocked by flutamide, indicating that testosterone may be responsible for the expression of TGF-₁ in mammary glands. TGF- RI and TGF- RII were also expressed strongly in testosterone-treated mammary epithelial cells and only weakly detectable in 17-estradiol treated and control mammary epithelial cells. Furthermore, TGF- RI and TGF- RII were also expressed in stromal cells, both in mammary tumors and in hormone-treated mammary glands. These observations indicate that the mechanism of testosterone in mammary carcinogenesis may be through its regulation of expression of TGF-₁ and its receptors. On the other hand, TGF- was also expressed in all 39 mammary cancers, while only 81% of the cancers were EGF-R positive. TGF- was also strongly expressed in stromal cells in all three experimental groups, but only moderately expressed in epithelial cells when treated with a combination of testosterone and 17-estradiol. By contrast, EGF-R was strongly expressed in epithelial cells in the three experimental groups but negative in stromal cells. Flutamide or tamoxifen was unable to block the expression of TGF- induced by the combined sex hormone treatment. However, they were effective in blocking the expression of TGF- when the animals were treated with testosterone or 17-estradiol alone, respectively. These results suggest that both testosterone and 17-estradiol may be required for the over-expression of TGF- in the mammary carcinogenesis induced by sex hormones. To our knowledge, this is the first experimental study to explore the regulation of TGF-₁, TGF-, and their receptors by testosterone and 17-estradiol in mammary carcinogenesis.     

Inhibitory effects of medroxyprogesterone acetate (MPA) and the pure antiestrogen EM-219 on estrone (E1)-stimulated growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat

Summary Estrogens are well known to play a predominant role in promoting the growth of DMBA-induced mammary tumors in the rat. Estrone (E₁), a steroid having weak estrogenic activity, is one of most important estrogens in post-menopausal women, where it is converted into the potent estrogen estradiol (E₂) by 17-hydroxysteroid dehydrogenase (17-HSD) in many peripheral tissues, including the mammary gland. In this report, we have studied the effect of a new antiestrogen (EM-219) (N-butyl, N-methyl-11-(3, 17-dihydroxy-17-ethinylestra-135(10), 14-tetraen-7-yl) undecanamide) on E₁-stimulated growth of DMBA-induced mammary tumors and compared its effect with that of medroxyprogesterone acetate (MPA) alone or in combination. After 18 days, ovariectomy (OVX) reduced total tumor area to 29.6 ± 7.1% of the original size, while E₁ (1.0 µg, twice daily) caused a 139 ± 21% increase in tumor size in OVX animals. MPA (1.5 mg, twice daily) partially reversed the stimulatory effect of E₁ to 66.0 ± 9.0%, while the antiestrogen EM-219 (40 µg, twice daily) decreased tumor size to 70.0 ± 10%. Combination of these two compounds led to a further inhibition of tumor size to 30.7 ± 7.4% of the value found in OVX animals treated with E₁. Tumor E₂ levels decreased from 1688 ± 155 pmoles/kg tissue in OVX animals receiving E₁ to 709 ± 92, 1347 ± 98, and 184 ± 11 pmoles/kg tissue in MPA-, EM-219-, and MPA + EM-219-treated OVX-E₁ animals, respectively. Treatment of OVX animals with E₁ increased by 69% the reductive activity of 17-hydroxysteroid dehydrogenase (17-HSD) while MPA abolished completely this effect of E₁. In the oxidative direction, treatment with E₁, E₁ + MPA, or E₁ + EM-219 had minimal or no significant effect on the activity of 17-HSD (vs OVX), while the combined treatment with MPA + EM-219 induced a 2-fold increase in 17-HSD activity, thus leading to an increased conversion of E₂ into E₁. The present data show that combination of the pure antiestrogen EM-219 with MPA exerts a greater reduction in DMBA-induced mammary tumor growth and intratumoral E₂ levels stimulated by E₁ than either compound used alone. This interactive effect of the antiestrogen and MPA could at least partially be related to the increased inactivation of E₂ into E₁. The present data suggest that such a combination could be a useful approach for the treatment of breast cancer, especially in post-menopausal women.     

Glioblastoma Multiforme in an Asian Population: Evidence for a Distinct Genetic Pathway

In Singapore astrocytic tumours occur in only 25% of patients with primary brain tumours compared to 40–60% in other series. Glioblastoma multiforme arises either de novo as a primary glioblastomas associated with epidermal growth factor receptor (EGFR) and mdm2 over-expression or as a secondary glioblastomas, through malignant progression from low-grade astrocytomas, associated with p53 mutations and PDGFR- over-expression. Using immunohistochemical methods and DNA sequencing, we studied our population of glioblastomas for over-expression of EGFR, mdm2, p53, and PDGFR- as well directly for mutations of the p53 gene. While levels of over-expression of EGFR and mdm2 were consistent with levels expected for primary glioblastomas, levels of p53 and PDGFR- were consistent with levels documented for secondary glioblastomas. Notably 96% of the samples over-expressed p53 as detected with monoclonal antibody pAb 240. Of the 39 samples available for DNA sequencing 18% (7/39) had p53 mutations, including three mutations previously undocumented in glioblastomas. These results provide strong evidence that glioblastomas in Asian patients do not conform to currently accepted models of glioblastoma development, and that clinically defined glioblastomas in these patients show genetic changes consistent with both primary and 'secondary glioblastomas.     

Genetic polymorphisms of <Emphasis Type="Italic">TGF-β</Emphasis>1 &; <Emphasis Type="Italic">TNF-β</Emphasis> and breast cancer risk

Objective. The proliferation of malignant breast epithelial cells is regulated by various stimuli including cytokines and growth factors, thus the variants of those genes may modify the breast cancer risk. To evaluate the potential influences of TGF-1 T²⁹C and TNF- A²⁵²G gene polymorphisms on breast cancer risk, a case–control study was conducted in Korea.Methods. Histologically confirmed breast cancer cases (n = 560) and controls (n=509) with no previous history of cancer were recruited from three teaching hospitals in Seoul, Korea. Genotypes were determined by PCR-CTPP (polymerase chain reaction with confronting two-pair primers) method. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by unconditional logistic regression model adjusting for age, body mass index, education, parity, age at first full-term pregnancy, and family history of breast cancer. Results. The TGF-1 ²⁹C-allele containing genotypes posed an increased risk of breast cancer (OR=1.3, 95% CI=1.02–1.79), especially in postmenopausal women (OR=1.6, 95% CI=1.01–2.44). Similarly, the TNF- ²⁵²G-allele containing genotypes posed an increased risk of postmenopausal breast cancer (OR=1.7, 95% CI=1.09–2.55). The risk of postmenopausal breast cancer increased in parallel with the number of the risk genotypes (p for trend <0.01). When data were stratified by the presumed non-genetic risk factors, TGF-1 C-allele containing genotypes were found to increase breast cancer risk almost two-fold in postmenopausal women with greater than median body mass index (>22.8 kg/m²) (OR=1.9, 95% CI=1.04–3.37).Conclusion. The results of this study therefore suggest that polymorphisms of TGF-1 and TNF- genes may modify individual susceptibility to breast cancer in Korean women.     

TM-1 cells from an established human malignant glioma cell line produce PDGF,TGF-α, and TGF-β which cooperatively play a stimulatory role for an autocrine growth promotion

Summary We have previously established a human malignant glioma cell line, TM-1. TM-1 cells could proliferate in the serum-free medium. In the present study, immunochemical analysis demonstrated that platelet-derived growth factor (PDGF), transforming growth factor (TGF)-, and TGF- are present in the serum-free medium conditioned by growing TM-1 cells. While the cells appeared to possess a single type of binding sites for epidermal growth factor (EGF) with properties comparable to those determined for other tumor cells, the conditioned medium did not contain EGF. PDGF, TGF-, and EGF added exogenously to serum-free media stimulated thymidine incorporation into DNA of TM-1 cells. In addition, antibodies specific for PDGF and TGF- suppressed this activity. These results indicate autocrine and stimulatory roles of PDGF and TGF- for the proliferation of TM-1 cells. As observed for other tumor cells, TGF- by itself weakly suppressed thymidine incorporation by TM-1 cells. However, TGF- employed in combination with TGF- or EGF appeared to stimulate thymidine incorporation, suggesting that a cooperative action of TGF- with different growth factors may be involved in the stimulatory growth regulation at least for TM-1 cells.     

The effects of TGF-α and 17β-estradiol on polyphosphoinositide metabolism in MCF-7 breast cancer cells

Summary The mechanism by which transforming growth factor-alpha (TGF-) stimulates breast cancer cell proliferation is largely unknown. Furthermore, its potential role as an autocrine effector of estradiol-17 (E₂)-stimulated growth of hormone-dependent mammary tumors remains controversial. Transient changes in phosphatidylinositol (PI) turnover have been demonstrated in several tissues in response to growth factors. In these experiments, we tested the effects of TGF- and E₂ on PI metabolism in three MCF-7 breast cancer cell sublines (MCF-7B, MCF-7I, and MCF-7J). Although TGF- was mitogenic in MCF-7I and MCF-7J cells, PI hydrolysis was stimulated by the growth factor only in the MCF-7I cells. In addition, the TGF- effect was relatively modest, ranging from 23% to 42%. E₂ effects on PI turnover were tested in the MCF-7B cells, which were the most sensitive to the proliferative effect of the hormone. E₂ did not stimulate PI hydrolysis, whether or not the cells were labelled in the presence of the hormone. On the other hand, E₂ did seem to stimulatede novo synthesis of phosphatidylinositol and induce activation of PI kinases. These results demonstrate that TGF--stimulated PI hydrolysis is modest and cell type dependent. At least under certain conditions, PI metabolism is not involved in the proliferative effects of TGF- (MCF-7J) or E₂ (MCF-7B). The role of increased PI synthesis in E₂-stimulated MCF-7 cell growth remains to be established.This work is supported by a grant from the National Cancer Institute, POI CA40011.     

Suppression of TGF-β1 in Human Gliomas by Retroviral Gene Transfection Enhances Susceptibility to LAK Cells

Human glioma cell line, Onda 10 produces TGF-1. TGF-1 has a biological role for the immunosuppression of the host. We have investigated whether suppression of TGF-1 on human glioma cell enhanced the susceptibility to lymphokine-activated killer (LAK) cells. In vitro, susceptibility to LAK cells on Onda 10 cell is augmented by retroviral gene transfection with antisense TGF-1. Nude mice bearing Onda 10 cells transduced with antisense TGF-1 gene has a longer life span compared to mice carrying that of sense TGF-1 gene or vector alone. The cytotoxic activity of LAK cells induced from spleen cells of mice carrying antisense TGF-1 gene transduced cells is higher against Onda 10 cell than that of LAK cells from mice carrying vector alone transduced cells. Also, antisense TGF-1 gene transduced cells are much more sensitive to LAK cells compared to Onda 10. These suggest that the augmented host systemic immunity in mice is one of the mechanisms of the reduced tumorigenicity of antisense TGF-1 gene transduced cells and that the increased systemic immunity could be ascribed to the increased immunogenicity of the tumor cells. The gene therapy for malignant glioma with antisense TGF-1 gene is expected to be promising.     

Interferon effect on glycosaminoglycans in mouse gliomain vitro

Summary The effect of mouse interferon / (MuIFN /) on the production of glycosaminoglycans (GAGs) by mouse glioma G-26in vitro was evaluated. Two GAG species secreted extracellularly by the mouse glioma G-26 were isolated using cellulose acetate electrophoresis. They were identified as hyaluronic acid (HA) and chondroitin sulfate (CS) following enzymatic digestion with enzymes: hyaluronidase and chondroitinase ABC. Further characterization of CS by enzymatic digestion with specific chondroitinases for chondroitin 4-sulfate (CSA) and chondroitin 6-sulfate (CSC), revealed that the isolated CS was neither CSA nor CSC. Therefore, it may be either chondroitin sulfate B (CSB) (dermatan sulfate) or one of the chontroitin sulfate isomers (D-H).The three day incubation of glioma G-26 cells with 8×10-8×104 U/ml of MuIFN / resulted in a dose dependent inhibition of cell proliferation measured by³H-thymidine incorporation and the MTT assay. The significant decrease of the CS (p < 0.008) but not the HA level, (measured densitometrically), was observed following 72 hours (hrs) incubation of G-26 cells with 8 × 10³ U/ml of MuIFN / (IFN treated cells: 0.03 ± 0.007 integrated optical density (IOD); control cells: 0.07 ± 0.01 IOD). The decreased CS production may be the underlying cause of IFN mediated inhibition of glioma cell proliferation.     

Bone marrow fibroblastic progenitors in patients with advanced breast cancer

Bone marrow fibroblast colony-forming cells (CFU-F) were studiedin fifteen consecutive untreated breast cancer patients (BCP)with clinical stages III and IV, and insixteen normal controls (NC). A decreased number ofCFU-F was observed in BCP compared to NC(p < 0.004). Confluence of the adherent celllayer was observed in all normal bone marrowmononuclear cells (MC) cultures, while a lower proportionof cultures from BCP (11/15) showed confluent adherentcell layers. When MC cultures of BCP weretreated with indomethacin (Indo, 10^-6M) 50% of themincreased the number of CFU-F compared to thevalue obtained without treatment. In addition, a significantincrease in the release of PGE2 in BCPcultures was observed before Indo treatment. Moreover, afterMC were fractionated into adherent and non-adherent progenitors,the CFU-F decreased in both types of fractionsof BCP compared to NC value (p <0.02 and < 0.05, respectively). The number oflight density MC per 10 ml of bonemarrow aspirate and the number of trypsin-sensitive adherentprogenitors were lower than NC in BCP (p< 0.02 and 0.013, respectively). Total MC andfibroblasts (fourth passage) were cultivated to evaluate theproduction of interleukin-1 (IL-1) by ELISA methodology. Resultsindicated no difference of IL-1 spontaneous release whentotal MC cultures of both groups were compared.However, the levels of this cytokine were lower(< 10 pg/ml) in fibroblast culture supernatants ofBCP compared to NC (1,217 ± 74 pg/ml).Fibroblast cultures from BCP showed low or norelease of IL-1 after muramyl-dipeptide (MDP, 1 µg/ml)stimulation. In conclusion, the defective proliferative and confluencecapacity of BCP fibroblastic progenitors may be relatedto the decrease in the production of IL-1by these precursors.     

Expression of a Soluble Transforming Growth Factor-β (TGFβ) receptor reduces tumorigenicity by regulating natural killer (NK) cell activity against 9L gliosarcoma in vivo

Immunotherapy of gliomas has been forwarded as an attractive alternative to standard therapeutic modalities. Numerous observations indicate some therapeutic efficacy with this approach, but it is not curative in most reports. It is well established that gliomas suppress immune reactivity via a number of mechanisms, including expression CD95 ligand (CD95L), which induces apoptosis of immune effector cells, and secretion of immunosuppressive factors such as transforming growth factor-beta (TGF). It has been hypothesized that abrogation of production or function of TGF would improve immune reactivity to gliomas. To investigate this in a fashion that is translatable into clinical practice, we utilized a retroviral vector encoding a truncated, soluble form of the Type II receptor for TGF (TFGsr) and expressed it in the rat 9L gliosarcoma line (9L-TGFsr). We then determined whether expression of TGFsr affected in vitro sensitivity of 9L to lysis by immune effector cells, whether expression of TGFsr affected tumorigenesis of 9L in vivo, and whether TGFsr affected expression of immunity to 9L. In these experiments, we determined that 9L-TGFsr was more susceptible than sham transfected 9L (9L-neo) to lysis by natural killer (NK) cells. We also determined that subcutaneously implanted 9L-TGFsr was less tumorigenic than 9L-neo in syngeneic rats. Similarly, survival was extended by 40% in rats given intracranial 9L-TGFsr compared to 9L-neo. Finally, we determined that elimination of CD161⁺ cells resulted in comparable growth of 9L-neo and 9L-TGFsr in vivo, indicating that NK or NK-like cells were responsible for the anti-tumor effects in this model.     

A novel approach of targeted ablation of mammary carcinoma cells through luteinizing hormone receptors using Hecate-CGbeta conjugate

Recent studies have shown that human and animal mammary gland carcinoma cell line express luteinizing hormone receptors (LHRs). We have examined the cytotoxic effect of Hecate-CG conjugate, that is, fusion of a lytic peptide (Hecate) and a 15-amino acid fragment of the CG-chain in vitro. To test the hypothesis that the Hecate-CG conjugate selectively abolishes cells possessing LHR, estrogen dependent and independent human breast cancer cell lines (MCF-7; MDA-MB-231) and a mouse Leydig tumor cell line (BLT-1) were treated in vitro with Hecate-CG conjugate and Hecate alone. Cytotoxic effects of the Hecate-CG conjugate and the Hecate alone was measured by lactate dehydrogenase (LDH) release immediately after treatment. We observed that the Hecate-CG conjugate selectively, in dose-dependent manner destroys cells possessing LHR in lower concentrations of preparate comparing to the Hecate alone and that the cytotoxic effect is strongly correlated with the number of LHR. Using Western blot analysis we characterized the LHR on membranes of MDA-MB-231, MCF-7 and BLT-1 tumor cell lines. In addition, we showed the evaluation of inhibition potential of the Hecate-CG conjugate to LHR. At a concentration of 33 µM the conjugate inhibited (50%; IC₅₀) the binding of CG to LHR.We suggest further development of this novel approach for the treatment of breast cancer by the Hecate-CG for in vivo trials.