Halogen-Substituted Derivatives of Dictyostelium Differentiation-Inducing Factor-1 Suppress Serum-Induced Cell Migration of Human Breast Cancer MDA-MB-231 Cells in Vitro

Triple-negative breast cancer (TNBC) is highly proliferative and metastatic, and because it lacks three major molecular targets for chemotherapy (estrogen receptor, progesterone receptor, and human epidermal receptor 2), it is extremely refractory. Differentiation-inducing factor 1 (DIF-1) and DIF-3, which are chlorinated alkylphenones, are lead anticancer compounds found in the cellular slime mold Dictyostelium discoideum. Here, we examined the in vitro effects of DIF-1, DIF-3, and 25 DIF derivatives on cell proliferation and serum-induced cell migration in human MDA-MB-231 cells, a model TNBC cell line. We found that Br-DIF-1, a chlorine-to-bromine-substituted derivative of DIF-1, strongly suppressed cell migration (IC50, 3.8 μM) with negligible effects on cell proliferation (IC50, >20 μM). We then synthesized 18 derivatives of Br-DIF-1 and examined the in vitro effects of these derivatives on cell proliferation and serum-induced cell migration in MDA-MB-231 cells. Among the derivatives, Br-DIF-1(+1), Br-DIF-1(+2), and Br-DIF-3(+2) exhibited strong anti-cell migration activities with IC50 values of 1.5, 1.0, and 3.1 μM, respectively, without affecting cell proliferation (IC50, >20 μM). These results suggest that these Br-DIF derivatives are good lead compounds for the development of anti-metastatic drugs against TNBC.


Introduction
Breast cancer is the most common cancer affecting women, accounting for 25% of all cancers in women [1,2]. Breast cancers are categorized according to histopathological type, grade, tumor stage, and receptor protein and gene expression [2][3][4][5]. For example, four subtypes of breast cancer categorized by receptor status are i) luminal A (estrogen receptor (ER) positive, progesterone receptor (PR) positive, and human epidermal receptor 2 (HER2) negative), ii) luminal B (ER-positive and/or PR-positive, and HER2-positive), iii) HER2-enriched (ER-negative, PR-negative, and HER2-positive), and iv) triple-negative (ER-negative, PR-negative, and HER2 negative) breast cancer [2]. Of these four In the present study, to screen for strong anti-metastatic compounds against TNBC, we examined the in vitro effects of DIF derivatives on serum-induced cell migration (SICM) in human MDA-MB-231 cells, a model TNBC cell line. We found that Br-DIF-1 strongly suppressed SICM without markedly affecting cell proliferation. We then synthesized 18 derivatives of Br-DIF-1 ( Figure 2) and examined the effects of the derivatives on SICM in MDA-MB-231 cells, and we found that several of the novel derivatives suppressed SICM more strongly than did Br-DIF-1 without markedly affecting cell proliferation. These results suggest that derivatives of Br-DIF-1 are good lead compounds for the development of anti-metastatic drugs against TNBC.

Cell migration Assay
Cell migration was assessed in vitro by using Trans-wells (#3422; Corning, New York, NY), as shown in Figure 3A. Trans-wells consist of two wells that fit inside one another, with the bottom portion of the upper well being a polycarbonate membrane containing micro-pores (pore size, 8 µm). MDA-MB-231 cells in DMEM-BSA (DMEM containing 4500 mg/mL of glucose supplemented with 100 units/mL of penicillin, 100 µg/mL of streptomycin, and 0.1% (w/v) bovine serum albumin) were added to the upper wells (5 × 10 4 cells/0.3 mL/upper well), while the lower wells were filled with 0.5 mL of DMEM-BSA (-FBS) or DMEM-FBS (+FBS); the media in both the upper and lower wells contained 0.1% (v/v) vehicle (DMSO or EtOH) or 1-10 µM of a DIF derivative. The upper wells were set on the lower wells, and then both wells were incubated for 16 h at 37 • C (5% CO 2 in air). After incubation, the cells in the upper wells were washed twice with phosphate-buffered saline, fixed for 3 min with methanol, and stained for 15 min with Victoria Blue solution (Nissui Seiyaku, Tokyo, Japan). After washing two more times with phosphate-buffered saline, the cells remaining in the upper well (i.e., those that did not migrate) were removed with cotton swabs, and the cells that had migrated to the lower well were counted by microscopic observation; four microscopic fields were examined per well and migration was assessed as the percentage of cells that had migrated toward the FBS relative to control.
To determine the 50% inhibitory concentration (IC 50 ) of each DIF derivative versus cell migration, the cell migration assay was performed in the presence of a range of concentrations of DIF derivatives, and IC 50 values were determined from the dose-response curves.

Cell Proliferation Assay
MDA-MB-231 cells (1 × 10 4 cells/mL/well) were incubated for 3 days at 37 • C (5% CO 2 in air) in 12-well plates with each well filled with 1 mL of DMEM-FBS. After the incubation medium was removed, the cells were washed with 1 mL of phosphate-buffered saline (pH 7.4) and incubated with 1 mL of fresh DMEM-FBS containing 5% (v/v) of the cell number indicator Alamar blue (Wako Pure Chemical Industries) until the color of the medium had changed. Relative cell number was determined by measuring absorbance at 570 nm (reference at 595 nm), as described previously [16,19].
To determine the 50% inhibitory concentration (IC 50 ) of each DIF derivative versus cell proliferation, the cell proliferation assay was performed in the presence of a range of concentrations of DIF derivatives, and IC 50 values were determined from the dose-response curves.

Statistical Analysis
Statistical analyses were performed by using Welch's t-test (two-tailed); a difference was considered significant when the P value was less than 0.05.

Effects of DIF Derivatives on Cell Migration and Cell Proliferation in MDA-MB-231 Cells
To investigate the structure-activity relationship of the DIF derivatives, we first examined the effects of the DIF derivatives (shown in Figure 1) on SICM in MDA-MB-231 cells in vitro ( Figure 3A-C). Among the DIF derivatives examined, Br-DIF-1 and Bu-DIF-3 at 5 µM most strongly suppressed SICM ( Figure 3B,C).
We then examined the effects of the DIF derivatives on cell proliferation in the MDA-MB-231 cells ( Figure 3D). Bu-DIF-3 most strongly suppressed cell proliferation, whereas Br-DIF-1 had no significant effect.

Comparison of the Biological Activities of Br-DIF-1 and Bu-DIF-3
To further assess the biological activities of Br-DIF-1 and Bu-DIF-3, IC 50 values relative to SICM and cell proliferation were determined in MDA-MB-231 cells and compared with those relative to LICM and cell proliferation in mouse osteosarcoma LM8 cells and relative to cell proliferation in mouse 3T3-L1 fibroblast cells, which are a model non-transformed cell line ( Figure 4, Table 1). Bu-DIF-3 strongly suppressed both cell migration and cell proliferation in all of the cell lines examined (Table 1), with IC 50 values at the micromolar level. Similarly, Br-DIF-1 also showed strong anti-cell migration activity in both MDA-MB-231 and LM8 cells with IC 50 values of 3.8 and 5.5 µM, respectively; however, its anti-proliferative activity was weak in MDA-MB-231, LM8, and 3T3-L1 cells (Table 1). Therefore, in subsequent experiments, we focused on Br-DIF-1 and its derivatives because we considered them to have the most potential for development as analytical reagents to investigate cancer cell migration or as anti-metastatic drugs.

Discussion
Metastasis is one of the most common causes of death in breast cancer patients [27,28]. The sites of metastasis of breast cancer include the lung, brain, and liver, but the most common site is bone; breast cancer patients with bone metastases have a poor prognosis [29][30][31][32]. Because TNBC is highly proliferative and metastatic and because molecular targeted therapies available for the treatment of other breast cancers are unavailable, TNBC is extremely refractory [4,[6][7][8][9]. Although investigations have been conducted to find novel chemotherapies for the treatment of TNBC [2,4,[33][34][35], new approaches to improve the current situation are still urgently needed.
In the present study, we found that Bu-DIF-3 strongly suppressed both SICM and cell proliferation in MDA-MB-231 cells (Table 1). We also have shown previously that Bu-DIF-3 suppresses cell proliferation and LICM in mouse osteosarcoma LM8 cells in vitro [21] (Table 1), and that Bu-DIF-3 suppresses serum-dependent cell migration (wound healing) in LM8 cells in vitro [36]. These results suggest that Bu-DIF-3 is good a lead compound for the development of anticancer agents that suppress both cancer cell proliferation and metastasis, including TNBC. However, given that Bu-DIF-3 suppresses cell proliferation in 3T3-L1 cells (a model of non-transformed cells) to almost the same extent as in cancer cells [21] (Table 1), it may induce side effects such as diarrhea and hair loss in vivo. In addition, anti-proliferative drugs usually inhibit the proliferation of both normal and cancer cells, which limits the dose that can be used therapeutically. Therefore, if a drug that specifically inhibits metastasis can be developed, an effective cancer treatment could be realized in combination with existing anticancer drugs. We therefore screened derivatives of Br-DIF-1, which we found to inhibit cell migration but not proliferation, to find potential lead compounds for development as anti-metastatic drugs. Among the Br-DIF-1 derivatives examined, we found that Br-DIF-1(+1), Br-DIF-1(+2), and Br-DIF-3(+2) strongly suppressed SICM, but not cell proliferation, in MDA-MB-231 cells in vitro ( Table 2). These results indicate that compounds with longer alkyl chains at the acyl group (e.g., Br-DIF-1(+1) and Br-DIF-1(+2)) have greater anti-cell migration activities than those with shorter alkyl chains. These results also suggest that Br-DIF derivatives could be useful tools for the analysis of cancer cell migration in vitro and good lead compounds for the development of anti-metastatic agents for use in vivo.

Conclusions
We examined the in vitro effects of Dictyostelium DIF-1 (a chlorinated polyketide) and its derivatives on SICM of human MDA-MB-231 cells, a model tripe negative breast cancer cell line. We found here that Br-DIF-1 (a chlorine-to-bromine substituted derivative of DIF-1) and several of its derivatives suppressed SICM strongly suppressed SICM without markedly affecting cell proliferation. The present results suggest that Br-DIF derivatives are promising lead compounds for the development of anti-metastatic drugs against TNBC.