Abstract
Morpholino-disiloxane (ALIS-409) and piperazino-disiloxane (ALIS-421) compounds were developed as inhibitors of multidrug resistance of various types of cancer cells. In the present study, the effects of ALIS-409 and ALIS-421 compounds were investigated on cancer promotion and on co-existence of tumor and normal cells. The two compounds were evaluated for their inhibitory effects on Epstein-Barr virus immediate-early antigen (EBV-EA) expression induced by tetradecanoyl-phorbol-acetate (TPA) in Raji cell cultures. The method is known as a primary screening test for antitumor effect, below the (IC50) concentration. ALIS-409 was more effective in inhibiting EBV-EA (100 μg/ml) and tumor promotion, than ALIS-421, in the concentration range up to 1000 μg/ml. However, neither of the compounds were able to reduce tumor promotion significantly, expressed as inhibition of TPA-induced tumor antigen activation. Based on the in vitro results, the two disiloxanes were investigated in vivo for their effects on mouse skin tumors in a two-stage mouse skin carcinogenesis study. The application of dimethyl-benzanthracene (DMBA; 390 nmol) as a tumor initiator was followed by exposure to TPA (1.7 nmol/l) as a tumor promoter. The experiments showed that ALIS-409 at a concentration of 85 nmol/l had a weak EBV-EA inhibitory effect in vitro and a moderate antitumor activity, compared to the positive control of DMBA plus TPA-treated mice. Flow cytometry by differential staining demonstrated interactions in co-cultures of MCF7 breast cancer and MRC5 human lung fibroblasts. The growth rate of tumor cells in mixed populations of MCF7 breast cancer and MRC5 normal fibroblast cells was reduced in the presence of ALIS-409, as compared to the control non-treated cell populations. The two disiloxanes were moderately-effective in chemoprevention in DMBA-induced and TPA-promoted in vivo tumor formation. Authors suggest that the inhibition of tumor cell and fibroblast interaction by ALIS409 might have some perspective in the development of anti-stromal therapy.
- Disiloxanes
- chemoprevention
- anti-promotion
- EBV-induced early antigen
- two-stage mouse skin carcinogenesis
- co-existence of tumor and normal cells
ALIS-409 and -421 are disiloxanes that were developed as modulators of multidrug resistance of various types of cancer cells. In the presence of ALIS-409 and -421, drug accumulation was increased in human multidrug-resistance gene-transfected mouse lymphoma cells and the Colo320-LRP cell line, but not in drug-sensitive human breast cancer lines MCF7 and T47D, and mouse lymphoma parental cells. The multidrug-resistant related protein (MRP)-mediated accumulation of carboxy-fluorescein in HTB.26/MRP human breast cancer cells and accumulation of daunorubicin in human 257P/MDR stomach cancer cells were not modified by these two compounds. A synergistic interaction between epirubicin and the two disiloxanes was found in mediated multidrug-resistance in the case of Colo-320/MDR1-LRP and mouse lymphoma cells only (1). The compounds were further investigated in vivo in an experimental mouse model of human pancreatic cancer xenograft (PZX-40/19G). The xenografted mice were treated with 10 mg/kg b.w. ALIS-409 every second day for 34 days. Some tumor growth retardation and increased apoptotic death of tumor cells was observed. In the treated group of animals, P-glycoprotein (P-gp) was expressed in 26% of tumor cells, whereas in the untreated tumors, 60% of cells displayed P-gp positivity. The multidrug-resistance reversal effect of ALIS-409 was demonstrated in vivo without any apparent toxicity (2). Furthermore, the vascular activity of the two disiloxanes were studied in endothelium-denuded rat aorta rings. The compounds antagonized 60 mM K+-induced contraction in a dose-dependent manner. Moreover, the inhibition of the L-type Ca2+ current in artery myocytes was concentration-dependent and the myorelaxant effect of the two compounds was attributed to the direct blockade of extracellular Ca2+ influx. This effect took place at much higher concentrations than at doses that modified multidrug-resistance of the cancer cells (3). The two ALIS derivatives eliminated plasmid-mediated tetracycline resistance from Escherichia coli cells and thereby inhibited antibiotic resistance (4). Additionally, the effect of both compounds on the killing activity of human macrophages infected with drug-resistant tuberculosis (XDR-TB) was determined by exposing the macrophages that had phagocytosed the bacterium to the ALIS compounds and assessing the killing activity by counting of colony-forming units. ALIS-421 was shown to have in vitro activity against XDR-TB and to transform non-killing macrophages into effective killers of phagocytosed XDR-TB without any cytotoxic activity at 3.5 μg/ml. ALIS-409 was less effective than ALIS-421 in this experiment (5).
Chemical structures of ALIS-409 and ALIS-421.
In another study, ALIS-409 was shown to modify the expression of glucose-regulated protein-78 (GRP78) and resistance to VP-16 in human NCI-H446 small cell lung cancer (SCLC) cells. The compound significantly reduced the overexpression of GRP78 induced by the Ca2+ ionophore A23187 and increased it chemosensitivity to VP-16 (6). Cytotoxicity of ALIS-421 was studied against a panel of human cancer cell lines, revealing cell-cycle arrest and apoptotic cell death in MDA-MB-435 breast cancer and HL-60 leukemia cells. Assessment of the global gene expression profile of ALIS-421-treated HL-60 cells was employed to identify cellular pathways affected by the compound and revealed disturbance of DNA replication, transcription and production of misfolded proteins. Endoplasmic reticulum stress and down-regulation of cell cycle, repair mechanisms and growth factor circuits resulted in induction of apoptosis. The reversal of taxane resistance may be linked to the down-regulation of gene expression of kinesins. The interference of ALIS-421 with DNA replication and transcription was suggested to be responsible for resistance, plasmid curing of microorganisms and selective toxicity on multidrug resistant cancer cells, thus defining a full range of putative cellular targets involved in anticancer and antimicrobial activity of ALIS-421 (7). The disiloxanes were synthetized as new, water-soluble organo-silicon compounds (8), which had strong inhibition of (ABC) transporters of multidrug-resistant cancer cells.
In the present study, ALIS-409 was investigated for its chemopreventive effects in vitro and in vivo. In addition, the model of tumor-stroma cell interaction was studied using co-cultures of human breast cancer cells and normal human fibroblasts. The effects of disiloxane ALIS 409 was studied on breast cancer cells and fibroblast interactions in a model of tumor-stroma relationships.
Materials and Methods
Compounds. Synthetic disiloxanes were patented with the names ALIS-409 and ALIS-421 (Figure 1) (European patent no. 1432717B1). In some papers they are mentioned as SILA-409 and SILA-421. These compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions at a concentration of 1.0 mg/ml. Other cell culture reagent, n-butyric acid was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). 12-O-Tetradecanoylphorbol-13-acetate (TPA) and 7,12-dimethylbenz[a]anthracene (DMBA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Animals. Specific pathogen-free female ICR mice (6-weeks-old) were obtained from Japan SLC Inc. (Hamamatsu, Japan) and were housed five per polycarbonate cage in a temperature-controlled room at 24±2°C with food and water ad libitum. All mice were fed Oriental MF (Oriental Yeast Co., Tokyo, Japan), ad libitum, during the experiments.
In vitro (EBV-EA) induction effect. The EBV genome-carrying lymphoblastoid cells, Raji cells Kanazawa, Japan), derived from Burkitt's lymphoma, were cultivated in RPMI-1640 medium. The Raji cells were incubated for 48 h at 37°C in a medium containing n-butyric acid (4 mmol), TPA (32 pmol) and different amounts of ALIS compounds. Smears were made from the cell suspension, and EBV-EA-inducing cells were stained by means of an indirect immunofluorescence technique. The details of the in vitro assay on EBV-EA induction have been reported previously (9-12).
In vivo two-stage carcinogenesis assay on the mouse skin papillomas promoted by TPA. The animals were divided into three experimental groups, each with 15 mice. The backs of the mice were shaved with surgical clippers, and they were treated topically with DMBA (100 μg, 390 nmol) in acetone (0.1 ml) as the tumor initiator. One week after initiation, papilloma formation was promoted twice-a-week by the application of TPA (1 μg, 1.7 nmol) in acetone (0.1 ml) to the skin. Group I received DMBA 30 nmol as negative control, Group II received DMBA + TPA as positive controls, not treated with ALIS. Group III, received topical application of ALIS-409 (85 nmol), in acetone (0.1 ml) respectively, one hour before the TPA treatment. The incidence and numbers of papillomas were monitored weekly for 20 weeks (13-16).
Cell cultures for co-cultivation. Normal and tumor cell lines were obtained from the American Tissue Culture Collection (ATCC) via LGC Promochem, Teddington, UK, and were used in the co-cultivation studies as follows. The drug-resistant sub-line of breast cancer MCF7 cells (MFC7/KCR) (ATCC: HTB22) was grown in RPMI-1640 medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum (FBS) in the presence of antibiotics (penicillin and streptomycin, Sigma, P4333).
Doxorubicin (1 μM) was added to the medium to maintain expression of P-gp.
Normal human lung fibroblast MRC-5 cells (ATCC: CCL-171) were cultivated in Eagle's (MEM) supplemented with 2 mM L-glutamine, 0.1 mM non-essential amino acids, 10% fetal bovine serum and antibiotics (penicillin and streptomycin Sigma, P4333).
Cell membrane labeling. PKH67 Green and PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich Ltd, Budapest, Hungary) was used for cell membrane labeling. PKH Fluorescent Cell Linker Kits provide the fluorescent labeling of live cells over an extended period of time, with no apparent toxic effects. The MCF7/KCR cells were stained by PKH67, and MRC5 fibroblasts were stained by PKH26.
Staining was performed according to the manufacturer's instructions. Briefly, the cells were washed with medium in the presence of serum. Ten million cells were added to a polypropylene tube, centrifuged, and carefully aspirated to maximize the removal of serum, but to minimize cell loss. Staining solutions of PKH67 and PKH26 were prepared in polypropylene tubes in diluent C (supplied with the kit) immediately prior to cell staining. Staining was initiated by rapidly adding a 2× concentrated cell suspension, prepared by resuspending the cell pellet in 1 ml of diluent C, to the 2× dye solution. Staining was stopped after 3 min by adding an equal volume (2 ml) of FBS over a period of 1 min and subsequently an equal volume of complete medium containing 10% FBS. The cells were then centrifuged and washed three times with 10 ml of complete medium. All steps were performed at room temperature. The cells were cultured in serum-free and in serum-supplemented medium for 24, 48 and 72 h for measurement of the distribution (MCF7/KCR – MRC5; cells prepared at 1:3; 1:10 and 1:30 ratios) of the mixed cell population. Stained cells were suspended with trypsin-EDTA and washed once in serum-free medium. The levels of green and red fluorescence of the cell populations were measured with a FACScan flow cytometer (Becton Dickinson, Oxford, UK). PKH67 is a green fluorochrome with excitation at 490 nm and emission at 504 nm, while PKH26 is a red fluorochrome with excitation at 551 nm and emission at 567 nm (17).
Results
The inhibitory effects on the induction of EBV-EA induced by TPA were examined for the ALIS compounds as a preliminary indicator of the capability of disiloxanes to inhibit the tumor-promoting activity of TPA. In detail, the TPA-induced EBV-EA (32 pmol TPA) activation in Raji cells was investigated in the presence of the two ALIS compounds. Even at a molar ratio of compounds: TPA of 1000:1, high Raji cell viability (60-70%) was observed indicating a low cytotoxicity of the test compounds. Both disiloxane compounds, ALIS-409 and ALIS-421 had an inhibitory effect, with a concentration for 50% inhibition with respect to the positive control (IC50) value of 240- to 487-fold the molar ratio/32 pmol TPA (Table I). Thus, these compounds were comparable to the reference compound, retinoic acid (IC50 482-fold molar ratio/32 pmol TPA) (Table I). Since the inhibitory effects of these two ALIS compounds against EBV-EA induction have been demonstrated to correlate with those against tumor promotion in vivo, these compounds can be considered as potential inhibitors of tumor promotion in two-stage carcinogenesis assays.
On the basis of these in vitro results, we subsequently evaluated the inhibitory effects of the two compounds in a mouse skin tumor model in vivo. The percentage incidence of papilloma in mice and the average numbers of papillomas per animal in the two-stage carcinogenesis test using DMBA as an inductor and TPA as a promoter are presented in Tables I and II, as well as in Figures 2, 3, 4 and 5. The incidence of papilloma in mice was high and reached 100% at 14-20 weeks promotion in the positive control group I. More than four and eight papillomas were observed per mouse at 11 and 20 weeks of promotion, respectively. The formation of papillomas in the treated mouse skin was delayed, and the mean numbers of papillomas per mouse were reduced by treatment with the ALIS compounds.
The effect of ALIS-409 on the model of stroma- tumor interaction was investigated using co-cultures of normal MRC5 and MCF7/KCR breast cancer cells in the presence and absence of bovine serum. In initial experiments, marked changes were found in the samples of co-cultured cells grown under the different conditions, revealing marked increases in the proportion of MCF7/KCR cells under serum-free conditions in the presence of MRC-5 fibroblasts (Figure 6). Similar results were obtained by flow cytometry when the breast cancer cells and MRC5 lung fibroblasts were mixed and incubated either in the presence or in the absence of serum in culture medium. ALIS-421 was applied at levels below the IC50 recorded in proliferative tests. The system was investigated in vitro by the double-staining method, using different ratios of MCF7/KCR breast cancer cells and MRC5 lung fibroblasts for the assessment of the effect of ALIS-421 on the two individual cell lines in co-cultures.
The relative activity ratios (%) of the ALIS compounds on activation of EBV-EA production.
Inhibitory effects of ALIS-409 on two-stage skin carcinogenesis in mice (10 mice used per group)
The ratio of MCF7/KCR tumor to MRC5 human fibroblasts during 24, 48 and 72 h of co-cultivation after seeding at a 1:3 ratio.
In order to attribute the changes in the co-cultures, clearly to the one or other cell line, the cells were labelled with two distinct PKH membrane linker dyes. The stained MCF7/KCR and MRC5 cells were mixed and cultured with or without serum at the ratio of 1:3. The changes in fluorescence intensities and fluorescence maximum of the two dyes were analyzed by flow cytometry after incubation for 24, 48 and 2 h and the proliferation of the two co-cultured cell populations was compared. As shown in Figures 6 and 7, the difference in the proliferation rates of the two cell types was minimal in the presence of serum. Under serum-free conditions, the ratio of tumor cells to normal cells was found to be higher than for the serum-supplemented co-cultures, exhibiting a drastic shift in favour of the MCF7/KCR tumor cell line.
As a control, MRC5 lung fibroblasts grew well in the presence of serum (Figure 6). The serum components serve as growth factors for both normal and cancer cells. In the absence of serum normal cells cannot grow, whereas cancer cells can feed on the normal cells (Figures 6 and 7) in vitro. Apparently, an interaction is formed in the complex tumor-normal cell relationship during tumor growth in the presence of ALIS-409 at 2 μg/ml. The concentration of the ALIS-409 was far below the 50% cytotoxicity level in the nutrient when MCF7/KCR and MRC5 cells were mixed in 1:3 ratio and cultured.
Flow cytometric analyses of the mixed cultures were performed after incubation for 24, 48 and 72 h in tissue cultures. The changes in the numbers of different cells in the mixed cell populations are shown in Table III. Noteworthy changes were found in the samples grown at a tumor cell:fibroblast ratio of 1:3 (Table III). The normal cells were more sensitive to serum starvation than were cancer cells. Without addition of serum to the culture medium, a tumor cell growth-inhibitory effect was found after incubation for 24 h in the presence of ALIS-409, which moderately reduced the proportion of MCF7/KCR cells. The difference in the effect of the ALIS-409 was higher after incubation for 72 h. The fluorescence intensity was initially low for MCF7/KCR cells, while after 48 h, the maximum fluorescence of the MCF7/KCR cells was increased due to the uptake of labelled compounds from normal cells (Figures 6, 7). The great decrease in the number of cancer and normal cells is apparent in the absence of serum, but the moderate increase (right shift) in the fluorescence maximum of the MCF7/KCR cell population still refers to the uptake of the labeled components of normal MRC5 fibroblasts by MCF7 cells in flow cytometric studies.
The inhibitory effects of ALIS-421 and ALIS-409 on two-stage skin carcinogenesis in mice. A. The number of mice with papillomas (%) weeks after promotion. B. Papillomas/mice weeks after promotion.
Mice of the group for the negative control. (DMBA was applied in 390 nmol without (TPA) promotion. Mice treated with DMBA 390 nmol + acetone. Pictures were taken after 20 weeks.
The positive control of two-stage skin carcinogenesis. Positive controls treated with DMBA 390 nmol + TPA 1.7nmol, picture was taken after 20 weeks.
The inhibitory effect of ALIS-409 on two-stage mouse skin carcinogenesis. Mice were treated with DMBA 390 nmol + TPA 1.7 nmol + Alis-409. Picture was taken after 20 weeks.
The growth rate of MCF7/KCR human breast cancer cells and MRC5 normal human fibroblast cells in the presence of bovine serum after 48 h incubation. Curve 1 shows the distribution of cells in the population of MCF7/KCR cells as control. Curve 2 shows the distribution of cells in the population of MRC5 cells in the control sample. Curve 3 illustrates the right shift fluorescence of cancer cells in the presence of normal cells in the mixed population showing an increased fluorescence due to the uptake of labeled MRC5 cells or their debris. The Figure also shows a remarkable decrease in the number of fibroblasts in the mixed culture comparing to the cancer cells.
Discussion
The carcinogenic activity of aromatic compounds is related to their electron acceptor properties (14). Anthracenes and benzanthracenes can induce oncogenes or modify the expression anti-oncogenes. Both biological effects result in cancer, i.e. in tumor formation. Electron-donating compounds are able to reduce cancer induction and reduce the effects of TPA as a tumor promoter. Proto-oncogenes in cells become overactivated, resulting in an overexpression of oncogenes (14). The amplification of oncogenes can be responsible for carcinogenesis. ALIS compounds can bind as electron donors to the cell membrane and possibly to DNA, and this binding may result in de-stabilization of oncogenes in chromosomes. Electron donor phenothiazines and ALIS compounds can operate as semi-conductors or can form different charge transfer complexes (CTCs) in the membrane (15), and substituted derivatives allow formation of such CTCs (23).
The growth rate of MCF7/KCR human breast cancer cells and MRC5 normal human fibroblasts in the absence of bovine serum after 48 h incubation. Curve 1: MCF7/KCR cells. Curve 2: MRC5 signed with PKH 67 green fluorescence. Curve 3: Mixed cell population (at initial 1:3 ratio).
The binding of SILA compounds as electron donors to biomembranes can generate conformational solitons, where the CTC can behave as intrinsic semiconductor, due to internal charge transfer. As a consequence of CTC formation in one direction via semi-conductor polypeptide, a conformational change can be induced in the target molecule that results in altered functions in tumor suppressor genes or de-localization of electrons or holes on the drug binding sites (15, 16).
Finally the formed CTC results in moderate chemo-prevention. The signal transfer via charge transfer in biological membranes is artificially changed during chemotherapy and well-studied in experimental chemotherapy in tumor–stroma interaction. In the case of the anti-TPA tumor-promotory effects of ALIS compounds, we can suppose that the ALIS compounds bind to biological membranes as electron donors and this binding generates a conformational soliton, where the CTC can behave as an intrinsic semi-conductor, due to internal charge transfer (15).
The tumor stroma is a multiplex cell population, which mediates tumor growth and invasion (18, 19). The parasitic co-existence of cancer and normal cells was inhibited in vitro (17). The inhibition of this cell–cell interaction seems to be a model for studies of the tumor and stroma interactions and the biological effect of ALIS-409 may lead to further research (17-19). A similar effect was found (20) when the effects of Foypan and ONO-3403 compounds were studied in co-culture of tumor and normal cells. The two compounds, namely the Foy-305 and ONO-3403 are used as anti-metastatic medicine in medical practice in Japan (21-22). Authors suggest that the ALIS compounds bind to various components of the cells by the formation of electron charge transfer complexes (23). Based upon the former studies and the results obtained some new perspective appears to find compounds for chemoprevention and inhibition of tumor-stroma interaction (24).
Acknowledgements
This study was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan (No.24300253), Szeged Foundation for Cancer Research (Szegedi Rákkutatásért Alapítvány) and the New Hungary Development Plan projects TÁMOP-4.2.2/B-10/1-2010-0012 and TÁMOP-4.2.2.A-11/1/KONV-2012-0035. The authors are also grateful to Dr. Imre Ocsovszki for and Aniko Váradi Vigyikán for the flow-cytometry measurements.
No conflict of interest on financial ties to discuss.
- Received February 14, 2013.
- Revision received March 23, 2013.
- Accepted March 27, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
References
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