Stearoyl-CoA desaturase 1 and paracrine diffusible signals have a major role in the promotion of breast cancer cell migration induced by cancer-associated fibroblasts.

BACKGROUND: Despite the recognised contribution of the stroma to breast cancer development and progression, the effective targeting of the tumor microenvironment remains a challenge to be addressed. We previously reported that normal fibroblasts (NFs) and, notably, breast cancer-associated fibroblasts (CAFs) induced epithelial-to-mesenchymal transition and increases in cell membrane fluidity and migration in well- (MCF-7) and poorly-differentiated (MDA-MB-231) breast cancer cells. This study was designed to better define the role played, especially by CAFs, in promoting breast tumor cell migration. METHODS: Fibroblast/breast cancer cell co-cultures were set up to investigate the influence of NFs and CAFs on gene and protein expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, as well as on the protein level and activity of its transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), in MCF-7 and MDA-MB-231 cells. To assess the role of SREBP1 in the regulation of SCD1 expression, the desaturase levels were also determined in tumor cells treated with an SREBP1 inhibitor. Migration was evaluated by wound-healing assay in SCD1-inhibited (by small-interfering RNA (siRNA) or pharmacologically) cancer cells and the effect of CAF-conditioned medium was also assessed. To define the role of stroma-derived signals in cancer cell migration speed, cell-tracking analysis was performed in the presence of neutralising antibodies to hepatocyte growth factor, transforming growth factor-ß or basic fibroblast growth factor. RESULTS: A two to three fold increase in SCD1 mRNA and protein expression has been induced, particularly by CAFs, in the two cancer cell lines that appear to be dependent on SREBP1 activity in MCF-7 but not in MDA-MB-231 cells. Both siRNA-mediated and pharmacological inhibition of SCD1 impaired tumor cells migration, also when promoted by CAF-released soluble factors. Fibroblast-triggered increase in cancer cell migration speed was markedly reduced or abolished by neutralising the above growth factors. CONCLUSION: These results provide further insights in understanding the role of CAFs in promoting tumor cell migration, which may help to design new stroma-based therapeutic strategies.

Recombinant human IFN-beta affects androgen receptor level, neuroendocrine differentiation, cell adhesion, and motility in prostate cancer cells.

We provide evidence that recombinant human interferon-beta (rHuIFN-beta) is able to increase androgen receptor (AR) expression, interfere with the acquisition of a neuroendocrine (NE) phenotype, and improve adhesion potential of androgen-insensitive prostate cancer cells (PC-3). The effect of rHuIFN-beta (10-1000 IU/mL) on AR, chromogranin A (CgA), E-cadherin (E-cad), N-cadherin (N-cad), and c-met levels was investigated by Western blotting after 48, 96, and 144 h. In agreement with our previous results, rHuIFN-beta (10-1000 IU/mL) induced a dramatic increase in AR (up to 5.3-fold, p < 0.001) that was already evident with the lowest cytokine concentration (10 IU/mL). A reduction in CgA levels (up to 45%, p < 0.002) was produced by 100 and 1000 IU/mL after 48-144 h. E-cad upregulation (up to 90%, p < 0.05) was observed starting from 96 h of treatment with 100 and 1000 IU/mL rHuIFN-beta and persisted until 144 h. An rHuIFN-beta-dependent reduction occurred in N-cad and c-met signal after a 48-96 h of treatment. This effect was particularly strong after 144 h of exposure to 1000 IU/mL rHuIFN-beta (81.5%, N-cad; 58%, c-met) (p < 0.002). Reverse transcription-PCR (RT-PCR) analysis of c-met expression demonstrated that the IFN-induced c-met downregulation mostly occurs at the transcriptional level (reduction up to nearly 50%, p < 0.000). Together, these results indicate that rHuIFN-beta may reduce the motility and invasiveness of poorly differentiated prostate cancer cells and interfere with the acquisition of an NE phenotype, often characterized by a low AR level.

[Effect of an LH-RH analogue on adhesion molecules in human prostate cancer cells].

UNLABELLED: BACKGROUND. The reduction in or the loss of the cell-cell adhesion often characterizes epithelial tumor initiation and progression. In the present study, we investigated the effects of the LH-RH analogue Leuprorelin acetate (LA), alone or associated with Dihydrotestosterone (DHT), on the expression of the adhesion proteins E-cadherin, α-, ß- and γ-catenin in androgen-sensitive (LNCaP) and -insensitive (PC-3 and DU-145) prostate cancer cells. MATERIALS AND METHODS. Protein expression was evaluated by Western blotting on cells treated for 48 h with LA (10-11 or 10-6 M) and 10-9 M DHT, alone or combined. RESULTS. In LNCaP cells, all the above mentioned molecules are expressed. PC-3 cells lack α-catenin, while DU-145 cells only express ß- and γ-catenin. In both LNCaP and PC-3 cells two truncated forms (97 and 35 kDa) of E-cadherin are present other than the functional protein (120 kDa). In LNCaP cells, no significant changes in E-cadherin (120 and 97 kDa) level were produced by DHT, while the 35 kDa fragment was reduced by 34%. LA increased the full length E-cadherin (26-30%) as well as the two fragments (30-49%). The addition of DHT to LA significantly reduced the analogue-induced E-cadherin raising. In LNCaP cells ß- and γ-catenin were up-regulated either by DHT (24% and 20%, respectively) or LA (up to 18% and up to 40%, respectively), while the expression of α-catenin was not modified. The combined DHT/LA treatment results in a less marked increase in ß- and γ-catenin levels. In PC-3 cells no changes in adhesion molecule expression were produced by LA treatment, while in DU-145 cells the analogue determined an appreciable reduction in ß- (20%) and γ-catenin (up to 35%) levels. CONCLUSIONS: The up-regulation of E-cadherin, ß- and γ-catenin in LNCaP cells by LA may be considered as another feature of the direct antitumor LH-RH analogue activity, as it may contribute to the maintenance/restoration of the normal architecture of prostate epithelium. The LA-induced modifications of catenins in DU-145 cells are worth some further investigations.

Apoptosis-related gene expression in benign prostatic hyperplasia and prostate carcinoma.

BACKGROUND: The aim of this study was to examine the expressions of the bcl-2, bax, fas and c-myc apoptosis-related genes in benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) to determine whether significant differences exist within each disease and between the two groups of patients. The correlation between gene expression and tumour diameter, stage, Gleason score and serum PSA was also investigated. PATIENTS AND METHODS: Tissue specimens from 51 cases of BPH and 27 cases of CaP were examined for bcl-2, bax, fas and c-myc expression by reverse transcriptase-PCR (RT-PCR). RESULTS: In BPH, bcl-2 and bax gave the weakest signals (p < 0.001). In CaP, bcl-2 was the least expressed gene (p < 0.001). In both patient groups, fas and c-myc were the most highly expressed genes (p < 0.05). Both bcl-2 and bax were expressed at higher levels in CaP than in BPH (p < 0.02). The bcl-2/bax ratio was lower in CaP than in BPH (p < 0.001). Bcl-2 was more highly expressed in high Gleason grade (> 7) tumours (p < 0.05). In the BPH group, bax showed a positive relationship with fas (p < 0.01), while the bcl-2 level inversely correlated with that of c-myc (p < 0.05). CONCLUSION: Our data showed that all the apoptosis-related genes were expressed in both BPH and CaP. The stronger expression of bax and the lower bcl-2/bax ratio observed in CaP may suggest a pro-apoptotic stimulus, while the higher bcl-2 levels appear to counterbalance the tendency to cell death.

Direct effects of GnRH agonists in human hormone-sensitive endometrial cells.

The antiproliferative effect of two GnRH agonists (leuprorelin acetate and triptorelin), alone or combined with tamoxifen (TAM) or medroxyprogesterone acetate (MPA), on human estrogen-sensitive endometrial cancer cells (Ishikawa) was investigated. Although ineffective when tested alone in all the culture conditions used, both analogues counteracted or even suppressed the estrogen-stimulated growth of Ishikawa cells. The antiestrogenic effect of TAM or MPA was not modified by their association with high doses of the GnRH analogues, but low concentrations of triptorelin combined with MPA 10(-7) M determined a reduction in cell numbers which was greater than that obtained with the progestin or the analogue alone. In addition, analogue treatment prevented the estrogen-induced decrease in the level of estrogen receptors. Our data provide evidence that GnRH agonists can directly inhibit estrogen-stimulated endometrial cancer cell growth and suggest that they may interfere with steroid-receptor machinery.

Effect of placenta growth factor-1 on proliferation and release of nitric oxide, cyclic AMP and cyclic GMP in human epithelial cells expressing the FLT-1 receptor.

We investigated the effect of placenta growth factor-1 (P1GF-1) on cell growth and on the release of nitric oxide (NO), cyclic AMP (cAMP) and cyclic GMP (cGMP) in human malignant epithelial cells. A noteworthy increase in proliferation was induced in choriocarcinoma cells (BeWo) by P1GF-1 treatment, while breast cancer cells (CG-5) were minimally affected. Western blotting and immunocytochemistry demonstrated the expression of the P1GF-1 receptor fms-like tyrosine kinase-1 (Flt-1) in these models. NO was released in the BeWo culture medium as a result of P1GF-1 treatment, with maximal induction occurring after 6 h. Enhanced cAMP levels were observed after 80 min-6 h, while the amounts of cGMP produced were undetectable. In summary, PIGF-1 stimulates the proliferation of cell types that express Flt-1, other than endothelial cells. In BeWo cells, this effect is preceded by the induction of NO and cAMP as probable downstream effectors of Flt-1 activation.

Effect of leuprorelin acetate on cell growth and prostate-specific antigen gene expression in human prostatic cancer cells.

OBJECTIVES: We investigated modulation of cell growth and prostate-specific antigen (PSA) gene expression in prostatic cancer cells by the luteinizing hormone-releasing hormone analog (LH-RHa), leuprorelin acetate, alone or combined with other agents. METHODS: The effect of the analog on proliferation of both androgen-sensitive and -insensitive prostate cancer cells, maintained in different culture conditions, was evaluated by cell counts at various intervals of time. Basal expression of PSA gene and its variations were determined by a reverse transcriptase-polymerase chain reaction assay. RESULTS: LH-RHa is ineffective in regulating cell growth, when used alone in both hormone-sensitive and -insensitive cell lines. Nevertheless, it counteracts the stimulatory action of androgens on proliferation of LNCaP cells, which respond to low concentrations of dihydrotestosterone. Moreover, LH-RHa has an inhibitory effect on the mitogenic action of epidermal growth factor (EGF) in androgen-unresponsive PC-3 cells. The analog reduces PSA gene expression in both hormone-sensitive and -insensitive cells. Interestingly, it counteracts the gene expression induced by androgens in LNCaP cells and by EGF in PC-3 cells. CONCLUSIONS: These data show that LH-RHa may behave like a negative growth factor, which directly regulates cell growth and PSA gene expression. Moreover, our findings support the idea that growth factors may interfere with the androgen signalling pathway.

Effect of Leuprorelin Acetate on Cell Growth and Prostate-Specific Antigen Gene Expression in Human Prostatic Cancer Cells.

Objectives: We investigated modulation of cell growth and prostate-specific antigen (PSA) gene expression in prostatic cancer cells by the luteinizing hormone-releasing hormone analog (LH-RHa), leuprorelin acetate, alone or combined with other agents. Methods: The effect of the analog on proliferation of both androgen-sensitive and -insensitive prostate cancer cells, maintained in different culture conditions, was evaluated by cell counts at various intervals of time. Basal expression of PSA gene and its variations were determined by a reverse transcriptase-polymerase chain reaction assay. Results: LH-RHa is ineffective in regulating cell growth, when used alone in both hormone-sensitive and -insensitive cell lines. Nevertheless, it counteracts the stimulatory action of androgens on proliferation of LNCaP cells, which respond to low concentrations of dihydrotestosterone. Moreover, LH-RHa has an inhibitory effect on the mitogenic action of epidermal growth factor (EGF) in androgen-unresponsive PC-3 cells. The analog reduces PSA gene expression in both hormone-sensitive and -insensitive cells. Interestingly, it counteracts the gene expression induced by androgens in LNCaP cells and by EGF in PC-3 cells. Conclusions: These data show that LH-RHa may behave like a negative growth factor, which directly regulates cell growth and PSA gene expression. Moreover, our findings support the idea that growth factors may interfere with the androgen signalling pathway.

Sensitivity of human melanoma cells to oestrogens, tamoxifen and quercetin: is there any relationship with type I and II oestrogen binding site expression?

We investigated the effect of oestrogens, anti-oestrogens and flavonoids on the growth of a human melanoma cell line (SK-Mel-28) and, at the same time, the presence of both type I oestrogen receptors (ERs) and type II oestrogen binding sites (type II EBS) to gain a fuller picture of the relationship between melanoma cell proliferation and receptor status. 17beta-Oestradiol (E2) and the flavonoid quercetin (Q) produced a marked inhibition of proliferation, but only at the highest dose used (10(-5) M) and only when added daily to the medium. Diethylstilboestrol (DES) (10(-5) M) was effective in inhibiting cell growth when the medium was renewed every 3 days and produced a more pronounced reduction when added daily to the medium. Tamoxifen (TAM) inhibited cell proliferation at a dose starting from 10(-7) M when the medium was renewed every 3 days. When added daily to the medium, it did not induce a greater inhibitory effect and it was cytotoxic at 5 x 10(-6) M and 10(-5) M. The antiproliferative effect of E2, DES and Q did not seem to be dependent on their interaction with ERs, which were minimally detected in SK-Mel-28 in both immunocytochemical and biochemical assays. Our model revealed, through a biochemical assay, a large number of type II EBSs which could be involved in the anti-oestrogen action, but this does not exclude the involvement of other mechanisms. Finally, TAM (10(-5) M) appeared to reduce the activity of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase, an effect that could be interesting from the point of view of the therapeutic efficacy of alkylating agents.

Growth inhibition of fibroblasts from nasal polyps and normal skin by lysine acetylsalicylate.

Some authors have shown that lysine acetylsalicylate (LAS) may help prevent nasal polyp relapses. As some anti-inflammatory drugs have been found to regulate cell growth, we investigated the antiproliferative effect of LAS on fibroblasts derived from nasal polyps. Moreover, we studied the effect of LAS on the growth of fibroblasts derived from normal skin to determine whether the response was similar to that obtained in the above-mentioned cells. Fibroblasts were obtained from tissue samples of nasal polyps from two aspirin-tolerant and two aspirin-intolerant patients, and from the normal skin of a healthy donor. The cells were treated with LAS (20-2000 microg/ml of culture medium). Cell growth and viability were evaluated after 3 and 6 days of culture. LAS had a growth-inhibitory effect on cells independently of their derivation. A reduction in cell growth was seen at the concentrations of LAS tested, which correspond to those used in the local treatment of nasal polyposis.

Natural interferon-alpha activity in hormone-sensitive, hormone-resistant and autonomous human breast-cancer cell lines.

This study explored the activity of natural interferon-alpha (nIFN-alpha) in regulating cell growth of 6 breast-cancer cell lines. The anti-proliferative effect of the combination nIFN-alpha and tamoxifen (TAM) or medroxyprogesterone acetate (MPA) in CG-5 estrogen-sensitive mammary cancer cells was investigated, and the ability of nIFN-alpha to restore hormone-sensitivity in the MPA-resistant MCF-7 SK sub-line was examined. nIFN-alpha, at concentrations ranging from 10 to 1000 IU/ml, inhibited cell proliferation of all cell lines tested after 3 and 6 days of treatment. In particular, the highest concentration of the drug used was equally effective in hormone-sensitive and in hormone-insensitive cells. A 6-day pretreatment of CG-5 cells with nIFN-alpha, at the above-mentioned doses, sensitized them to the growth-inhibiting activity of subsequent exposure to 10(-7) M TAM or MPA, which resulted in a synergistic effect, and could be explained on the basis of the observed enhancement of estrogen and progesterone receptors due to IFN activity. Conversely, the simultaneous drug combination did not modify the response to the hormone in CG-5 cells. Pre-treatment with nIFN-alpha (from 10 to 1000 IU/ml) restored MPA sensitivity in the MCF-7 SK sub-line, but no modulation of progesterone receptors was seen in this model. The hormone-sensitivity of the parental cell line was not substantially affected by pre-exposure to nIFN-alpha. These data indicate that nIFN-alpha may be potentially useful in enhancing the clinical effectiveness of TAM and MPA and in overcoming hormone resistance.

Interferons inhibit EGF-stimulated cell growth and reduce EGF binding in human breast cancer cells.

CG-5 estrogen-sensitive human breast cancer cells contain specific Epidermal Growth Factor receptors (EGF-R, Kd = 0.09-0.17 nM) and respond to the mitogenic effect of EGF. The increase in cell proliferation has been observed starting with very low concentrations of EGF (10-12M) and was statistically significant at all doses. Nevertheless, cell growth stimulation was emphasized when cells were grown under stringent culture conditions. When cells were exposed to 100 IU/ml of natural beta-interferon (n beta-IFN) the binding of EGF to the cell membrane was reduced after 72 hours of treatment, while the exposure of CG-5 cells to 1000 IU/ml of n beta-IFN resulted in an EGF-R reduction which started after 48 hours and became statistically significant after 72-120 hours. If CG-5 cells were treated with 1000 IU/ml of recombinant alpha 2b-interferon (ra2b-IFN) this reduction was observed after 168 hours of exposure to the drug. Both the IFNs abolished EGF-stimulated cell growth. Our results indicate that IFN treatment down-regulates EGF-R in estrogen-sensitive breast cancer cells and suggests that this down-regulation may be involved in the inhibitory action of IFN on cell growth.

Interferon and hormone sensitivity of endocrine-related tumors.

Interferons (IFNs) have been shown to enhance both in vitro and in vivo the antiproliferative activity of some hormones and anti-hormones which mainly act via steroid receptors. We discuss some of the mechanisms which could be involved in determining this effect in breast, endometrial and prostatic cancer cells, with a particular emphasis on steroid receptor modulation, reduction of the expression of epidermal growth factor receptors and, finally, down-regulation of some oncogenes. It seems that under appropriate conditions IFN might produce changes in cancer cells that enhance or restore hormone sensitivity. Nevertheless, available clinical data are too few to allow any conclusion to be drawn and this problem merits further investigations.

Effects of interferon-beta on steroid receptors, prostaglandins and enzymatic activities in human endometrial cancer.

Steroid receptors, prostaglandin output and enzymatic activities were determined in explants derived from human endometrium exposed to natural interferon-beta (IFN-beta). Receptors and cell metabolism were evaluated before culturing the tissue fragments and after a 3-day treatment with varying concentrations of IFN-beta. Total steroid receptor levels were unchanged when explants were set up, but there was a redistribution of both estrogen and progesterone receptors (ER and PR). A decrease in cytoplasmic receptors corresponded to an increase in receptor molecules within the nucleus. Treatment with low concentrations of IFN-beta caused a significant enhancement (p < 0.05) of ER and PR in neoplastic endometrium. In basal conditions the ratio between prostaglandin F2 alpha (Pgf2 alpha) and prostaglandin E2 (PgE2) was higher in normal than in neoplastic endometrium. The addition of low concentrations of IFN-beta to the culture medium determined a significant increase (p < 0.02) in PgF2 alpha and a parallel increase in the above ratio in neoplastic tissue, while no variation was found in normal endometrium. Analysis of the results concerning the variations in hormone-related enzymatic activities due to IFN-B revealed a significant increase (p < 0.05) in 17 beta-hydroxy-steroid-dehydrogenase (17 beta-HSD) activity. The data presented here indicate that treatment with IFN-beta modifies those biological characteristics of neoplastic cells which are involved in hormone-responsiveness.

Direct antiproliferative effect of triptorelin on human breast cancer cells.

We investigated the effect of Triptorelin (Decapeptyl, DEC) alone or combined with Tamoxifen (TAM) or Medroxyprogesterone acetate (MPA) in human breast cancer cells. DEC did not affect the growth of estrogen-insensitive MDA-MB-231 cells, while it inhibited the estrogen-stimulated proliferation of MCF-7 and CG-5 cells. No amplification of growth inhibition induced by TAM or MPA was determined by DEC. Progesterone receptor levels of CG-5 cells were significantly enhanced by DEC in the presence of 17 beta-estradiol (E2) with respect to those in control and E2-treated cells.

Androgen receptors and hormone sensitivity of a human prostatic cancer cell line (PC-3) are modulated by natural beta-interferon.

Androgen receptors are expressed at a low level in the cell line PC-3, which does not respond to either androgens or antiandrogens. If these cells are exposed to natural beta-interferon (beta-IFN) a reduction in cell growth and an increase in androgen receptors, evaluated by both biochemical and immunocytochemical techniques, occur. This increase seems not to be related to a selective block of PC-3 in any phase of the cell cycle. Pretreatment with beta-IFN determines in PC-3 cells a partial responsiveness to the androgen dihydrotestosterone as reflected by the increase in cell number. Moreover, the antiandrogen hydroxyflutamide shows agonistic properties by increasing the cell number of PC-3 cells pre-exposed to beta-IFN. When the antiandrogen is tested in combination with interferon, it produces a reduction in the beta-IFN-induced inhibition of cell growth. It is not known whether these unexpected effects are due to the increase in androgen receptors or to other mechanisms.

Combined effects of estradiol, leuprorelin, tamoxifen and medroxyprogesterone acetate on cell growth and steroid hormone receptors in breast cancer cells.

Both tamoxifen and medroxyprogesterone acetate have a direct antitumor effect and are widely used in breast cancer therapy. Luteinizing-hormone-releasing hormone analogs inhibit the growth of breast cancer cells and could represent an alternative treatment for patients affected by breast cancer. Our study was carried out to investigate the effect of leuprorelin (TAP-144) alone or combined with tamoxifen or medroxyprogesterone acetate in human breast cancer cells. Ineffective when used in the absence of estrogens, TAP-144 inhibited the estrogen-stimulated growth of MCF-7, CG-5 and ZR-75-1 cells cultured in medium supplemented with charcoal-treated serum. The growth of estrogen-unresponsive MDA-MB-231 cells was not affected by TAP-144. The combination of TAP-144 with tamoxifen in CG-5 cells did not determine any enhancement of inhibition of cell growth, whereas in both CG-5 and MCF-7 cells, when 1 microM TAP-144 was associated with 0.1 microM medroxyprogesterone acetate, cell growth inhibition was increased, resulting in a subadditive effect. Progesterone receptor levels of CG-5 cells were significantly increased by TAP-144 in the presence of 17 beta-estradiol with respect to those present in control and 17 beta-estradiol-treated cells.

Natural interferon-beta treatment and steroid hormone receptors in primary endometrial cancer.

Forty-three patients with primary endometrial carcinoma were treated with natural interferon-beta (IFN-beta) at two different dose levels (2 x 10(6) IU or 6 x 10(6) IU im 3 times/week for 1 week). IFN-beta increased receptors for estrogens (ER) and progesterone (PR) in a high percentage of the 40 evaluable patients, without modifying the receptor affinity. The ER and PR enhancement, which was simultaneous in at least 50% of patients, and the increase of over 100 fmol/mg protein observed in some cases suggest that IFN-beta exerts a profound influence on receptor expression and, probably, on the hormone sensitivity of the tumor.

Steroid receptor enhancement by natural interferon-beta in advanced breast cancer.

In the current study we investigated the effect of two different doses of natural interferon-beta (IFN-beta) on steroid hormone receptors in 45 patients with advanced breast cancer. IFN-beta seems to regulate the receptor mechanisms, inducing in cutaneous metastases an increase of oestrogen and progesterone receptors. Moreover, using IFN-beta and tamoxifen as a combined therapy in 23 receptor-positive patients, no negative interference of the two drugs was observed and no relevant side-effects due to the treatment were noticed. The modulation of steroid receptor content by IFN-beta in advanced breast cancer might represent an interesting way to ameliorate the clinical responsiveness to anti-oestrogens.

Effect of natural beta-interferon on estrogen receptor mRNA of breast cancer cells.

We have demonstrated that natural beta-interferon (beta-IFN) enhances estrogen receptor (ER) mRNA of a human breast cancer cell line, CG-5. Cells were sensitive to the effect of beta-IFN at concentrations ranging from 10 to 100 IU/ml. The increase of ER mRNA was seen after 48 hr of treatment in at least three separate experiments. Our results are in agreement with the previously observed enhancement of receptor protein. In addition, they suggest that the IFN-induced promotion of the antiproliferative activity of drugs which act via ER may be due, in part, to increased receptor synthesis.