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Breast cancer is the second leading cause of cancer deaths in US women. We evaluated two novel compounds, piperidinyl-diethylstilbestrol (DES) and pyrrolidinyl-diethylstilbestrol (DES) for cytotoxicity against brine shrimp larvae, MCF-7 and rat normal liver cells.
In vivo cytotoxicity was evaluated against shrimp larvae for 24 h, while in vitro cell toxicity was evaluated by dye binding crystal-violet method after 48 h. The role of these compounds on different phases of the cell cycle was assessed by flow cytometry.
In shrimp assay, piperidinyl-DES and pyrrolidinyl-DES were potent with 50% effective dose (ED50) values of 7.9±0.38 and 15.6±1.3 μM, respectively. In MCF-7 and normal liver cells, the 50% lethal concentration (LC50) values were 19.7±0.95, 17.6±0.4 μM and 35.1 and >50 μM, respectively. Cell cycle analyses indicated that MCF-7 cells were arrested at the G0/G1 stage with these compounds.
The results indicate that pyrrolidinyl-DES possesses highly selective, potent anticancer activity.
Although extensive preclinical and clinical studies have resulted in modest success in decreasing the morbidity of breast cancer, to date no successful chemotherapeutic drug is available to control this disease. Every year, several thousands of new chemical entities undergo screening in various cell cultures but eventually do not achieve drug status due to severe side-effects. Ideally, one of the criteria for a drug being good is that it should not exhibit any undesirable side-effects on normal cells. In an effort to minimize the side- effects, the National Cancer Institute and others (1-10) have been exploring the vast resource of natural products for potent and selective anticancer agents but as yet, the success rate in discover such molecules has remained very low. Our earlier attempts to purify new compounds from plant sources have resulted in two novel anticancer molecules (8). One of these molecules was reported to exhibit selective anticancer activity in various cell cultures (6) but the subsequent preclinical studies with this compound in tumor-bearing nude mice did not yield good results due to its solubility problem (unpublished observations). As part of our ongoing drug discovery research in synthesizing potent and selective anticancer synthetic compounds, we identified two novel drugs, piperidinyl-DES and pyrrolidinyl-DES, and screened initially for lethality against brine shrimp larvae and extended the evaluations for cytotoxicity as well as antiproliferative potential by flow cytometry in the human estrogen-dependent MCF-7 breast carcinoma cell line. In addition, the cytotoxicity of these compounds was also evaluated in a normal rat liver cell line, CRL-1439, for comparison.
Trypsin, minimum essential medium, antibiotic-antimycotic solution (Ab/Am) and 4-hydroxy tamoxifen were purchased from Sigma Chemical Company (St. Louis, MO, USA). Fetal bovine serum, RPMI-1640 (modified), penicillin, streptomycin and L-glutamine were purchased from Media Tech (Herndon, VA, USA). Cosmic calf serum was purchased from Hyclone Lab Inc (Logan, UT, USA) and gentamicin reagent solution from Invitrogen (Carlsbad, CA, USA). Human insulin (Novolin R) was purchased locally. All other routine chemicals were of analytical grade.
In this technique, the in vivo potency of piperidinyl-DES and pyrrolidinyl-DES was evaluated against shrimp larvae. The assay was performed in triplicate vials at different doses (5, 10 and 15 μM) as described by Meyer et al. (11) and modified by McLaughlin (12). Shrimp larvae in ethanol (0.15%, v/v) in artificial seawater served as control. 4-Hydroxy tamoxifen (5, 10 and 15 μM) was used as a positive control. At the end of the incubation period, the surviving larvae per dose were counted and utilized for analysis.
Human estrogen-dependent breast carcinoma (MCF-7) cell line and normal rat liver cell line (CRL-1439) were purchased from the American Type Culture Collection (Rockville, MD, USA) and were used in the present investigation. These cell lines were maintained as described elsewhere (6, 13).
The cytotoxic studies with cell cultures were carried out in polystyrene, flat-bottom 96-well microtiter plates as described elsewhere (9). All test compounds were dissolved in ethanol at 10 mM stocks, from which working stocks were prepared in complete culture medium prior to treatments. Cells were treated with piperidinyl-DES or pyrrolidinyl-DES at different concentrations (5 to 100 μM). 4-Hydroxy tamoxifen (5 to 100 μM) was used as a positive control. The culture plates were incubated for 48 h continuously without further renewal of growth media in 5% CO2 at 37°C, with the plates capped in the normal fashion. All studies were repeated at least twice (n=8 to 12). After 48 h of exposure, the cytotoxicity of the compounds was evaluated by dye uptake assay using crystal violet as described elsewhere (2).
MCF-7 cells (0.18×106/ml) were treated with ethanol (0.15% , control), or piperidinyl-DES (5, 10 and 15 μM), or pyrrolidinyl-DES (5, 10 and 15 μM), or 4-hydroxy tamoxifen (5 μM) in triplicate dishes for 48 h in a 5% CO2 incubator at 37°C. Cells were fixed in 100% ice-cold ethanol for at least 24 h at 4°C. They were then stained with 1 ml of freshly prepared staining solution containing 50 μg/ml propidium iodide, 1.3 mg/ml ribonuclease A and 1 mg/ml D-glucose and incubated at room temperature for 1 h in the dark with occasional stirring. The proportion of cells in each stage was assayed within 2 h by using a FACSCalibur flow cytometer (Becton Dickerson, San Jose, CA, USA). In each sample, a total of 13,000 individual events from the gated subpopulation were analyzed separately. CellQuest software was used for acquisition and analysis of the data, and the percentage of cells in each phase was determined.
In the present study, the degree of selectivity of the synthetic compounds is expressed as per the previous report (14) with a minor modification (6): SI=LC50 of pure compound in a normal cell line/LC50 of the same pure compound in cancer cell line, where LC50 is the concentration required to kill 50% of the cell population.
The experimental results are presented as mean±standard error of the mean (SEM). The data were analyzed for significance by one-way ANOVA and then compared by Bonferronic multiple comparison tests using GraphPad Prism Software, Version 3.00 (San Diego, CA, USA). The LC50 and effective dose needed to kill 50% of shrimp larvae (ED50) were determined from the line graphs where both curves crossed (15). The test value of p<0.01 was considered highly significant.
Addition of piperidinyl-DES or pyrrolidinyl-DES (or 4-hydroxy tamoxifen) at 5, 10 or 15 μM to sea salt solution did not result in any precipitation. This is one of the primary requirements for an ideal compound for preclinical studies. It was observed that both piperidinyl-DES and pyrrolidinyl-DES caused significant (p<0.01) death of larvae at concentrations greater than 5 μM in a dose- dependent manner in comparison to the untreated control. Towards the end of incubation, the shrimp larvae in control vials were seen to be swimming actively, while the others in treated vials appeared very weak in terms of their swimming abilities. The ED50 values of piperidinyl-DES, pyrrolidinyl-DES and 4-hydroxy tamoxifen were (±SEM) 7.9±0.38 (3.2 μg/ml), 15.6±1.3 (6.3 μg/ml) and 9.2±0.7 μM (3.5 μg/ml), respectively (Table I).
Based on the high activity against the shrimp larvae, the in vitro anticancer activity of both test compounds was evaluated at different concentrations against human MCF-7 breast adenocarcinoma cells after 48 h of treatment. It was observed that in comparison to the control, both compounds caused significant (p<0.01) dose-dependent cell death to a similar extent. The LC50 values (±SEM) for piperidinyl-DES and pyrrolidinyl-DES were 19.7±0.95 μM (8.2 μg/ml) and 17.6±0.4 μM (7.1 μg/ml), respectively (Table I). 4-Hydroxy tamoxifen under our experimental conditions had an LC50 of 15.6±1.0 μM (6 μg/ml) on MCF-7 cells (Table I), but others (16) reported a much higher value (31 μM) for the same cell line. The reason for this could be due to differences in the assay methods (13).
When tested in a rat normal liver cell line, CRL-1439, both compounds exhibited significant differential cytotoxic activities (Table I). For example, the LC50 of piperidinyl-DES was 35.1±0.77 μM (14.6 μg/ml), while in the case of pyrrolidinyl-DES, it was >50 μM (>20 μg/ml). The LC50 of 4-hydroxy tamoxifen in the normal liver cells was 20.25±0.25 μM (7.8 μg/ml).
Figures 1 and and22 show the effects of increasing concentrations of piperidinyl-DES and pyrrolidinyl-DES on MCF-7 cell progression through G0/G1-, S- and G2/M-phases. It was observed that in comparison to the control, piperidinyl-DES and pyrrolidinyl-DES at different concentrations for 48 h resulted in cell cycle arrest significantly (p<0.01) in the G0/G1-phase and in the reduction of the number of cells in the S- and G2/M-phases. This demonstrates that both these synthetic compounds arrest cell cycle progression at the G0/G1 to S transition. The accumulation of cells at this phase was significant at 10 and 15 μM with both compounds (p<0.01), correlating with a subsequent significant decrease in S- and G2/M-phase cells at these concentrations (p<0.01). The positive control 4-hydroxy tamoxifen arrested the cells at the G0/G1-phase of the cell cycle.
The in vivo brine shrimp lethality is a simple bioassay considered as a useful tool for primary screening of various kinds of water-soluble compounds (2-7, 9, 11, 12). The significant difference (2-fold) in the ED50 values of both synthetic compounds here may suggest that the mode of action of these compounds in brine shrimp larvae were entirely different. Comparison of ED50 values (Table I) clearly shows that piperidinyl-DES was approximately 2-fold more toxic to shrimp larvae in comparison to pyrrolidinyl-DES (p<0.01). Based on the observation that the ED50 values of these compounds were less than 1,000 μg/ml, both were considered very active as per the earlier standard for potency of compounds in shrimp assay (11).
In the case of our in vitro studies with MCF-7 cells, the similarity of the LC50 values of piperidinyl-DES, pyrrolidinyl-DES and 4-hydroxy tamoxifen suggests that they may have the same target sites in these cells namely the estrogen receptor. This speculation, however, needs further studies for confirmation.
The significant difference in LC50 values of piperidinyl-DES and pyrrolidinyl-DES in the normal cell line reflects an interesting difference in the target sites in adenocarcinoma and normal cells. As per the earlier standard (17), pure compounds with potency of 4 μg/ml or less in cell culture studies are further considered for evaluation as chemotherapeutic agents in preclinical studies using animal models. In our study, in spite of slightly higher LC50 values of piperidinyl-DES and pyrrolidinyl-DES in MCF-7 cells in comparison to the earlier set standard for potency (17), the differential toxicity exhibited by these compounds is highly significant and may warrant further active investigation.
As the SI demonstrates the differential activity of a pure compound, the greater the SI value is, the more selective it is. An SI value less than 2 indicates general toxicity of the pure compound (14). Based on this, the SI data shown in Table I indicate that pyrrolidinyl-DES exhibits a high degree of cytotoxic selectivity. It is, however, unclear, as to why this compound exhibited a similar extent of toxicity in brine shrimp and MCF-7 cells. Interestingly, the SI for 4-hydroxy tamoxifen, which is widely used as an antiestrogen treatment for human breast cancer, was less than 2. This suggests its general toxicity to cells, as per the earlier standard (14).
Comparison of brine shrimp and cell culture assays (Table I) reveals that piperidinyl-DES shows a 2-fold differential toxicity in shrimp larvae as well as in MCF-7 cells, while this was not observed in the case of pyrrolidinyl-DES. The high degree of correlation between the cytotoxicity of the shrimp larvae and MCF-7 in this study is consistent with previous reports (7, 18) with different anticancer agents.
One of the important criteria for a therapeutic drug for cancer is to have minimum or no side-effects on normal body cells of patients undergoing chemotherapy. One way to achieve this is by employing lower doses of drugs. This invariably implies that the drug should not only have high potent activity at lower concentrations but also should exhibit high degree of selectivity. The present in vitro studies demonstrate the ability of the synthetic compound pyrrolidinyl-DES for high selective toxicity at lower concentrations (Table I). Since estrogens are associated with the etiology of human breast cancer, inhibition of estrogen-dependent cell growth by synthetic compounds such as pyrrolidinyl-DES, shown by cell cycle analysis in this study (Figures 1 and and2),2), may become a good therapeutic strategy to use as an antagonist for treatment of this dreaded disease. Further studies, however, are required to ascertain the antiestrogenic activity of pyrrolidinyl-DES. Current studies are under way to determine the efficacy of this compound as an anticancer activity in animal models.
This research project was supported by NCRR/RCMI G12RR03020, NIGMS/MBRS/SCORE GM08111, and HRSA SD34HP04018.