Breast cancer is the second most common neoplasm in women after skin cancer. In 2011, an estimated 288 000 individuals in the USA were diagnosed with breast cancer 1. Roughly 30% of all breast cancer patients will ultimately go on to develop metastatic disease and the majority of deaths from breast cancer are from recurrent or metastatic breast cancer 2.
Over the last decade, compelling evidence has been generated showing that the CXCR4/CXCL12 chemokine axis promotes tumor cell proliferation, invasion, and metastasis 3,4. It does this by promoting a tumor microenvironment that facilitates progression, angiogenesis, and invasion 5. The fact that over 20 different human tumor types have been documented to overexpress CXCR4 4,6–16 has made blocking the function of the CXCR4/CXCL12 axis an exceptionally important therapeutic target. In preclinical animal models, including human breast cancer xenografts, there is ample evidence that cancer metastasis can be inhibited by targeting the CXCR4 receptor 4,17–22.
Chemokines represent a signaling system that cancer cells use to metastasize to other sites within the body 23 and organs expressing high levels of CXCL12 can induce cancer cells overexpressing the CXCR4 receptor to metastasize to those specific sites.
Sites that normally express high levels of CXCL12 in the body are the bone marrow, lungs, lymph nodes, and liver; these sites are also the most common locations of breast cancer metastasis 6. Inhibition of CXCR4-dependent metastasis has been shown using antibodies 6,24, RNAi 25, siRNA 26,27, functional antagonist peptides such as T140 18, or other small peptides 22,28, small molecules such as AMD3100 17,21,24,25,29, and heparinoids 30. These compounds either bound to CXCR4, preventing CXCL12 from binding and activating the intracellular pathways that lead to metastasis, proliferation, and vascularization, or they inhibited the production of CXCR4 with the same results. In theory, either the CXCR4 receptor or the CXCL12 ligand could be targeted to inhibit tumor spread. However, it has been shown that inhibiting CXCL12 would be much more likely to interfere with the generation of antitumor immunity 3. Despite the number of different compounds being developed to block the effects of the CXCR4/CXCL12 axis on tumor metastasis, none have yet emerged with all the characteristics needed for effective antimetastatic cancer treatment. There is still a need for CXCR4 inhibitors with good bioavailability, long half-lives in the blood stream, and little, if any, toxicity.
Alexidine, a bisbiguanide similar in structure to the compounds synthesized for this study, has been reported previously to reduce cancer metastasis in mice 31. However, the cancer cells in this earlier study were pretreated with alexidine before an intravenous injection, likely resulting in some cell death and making interpretation of the results difficult. Therefore, alexidine was re-evaluated in this study using a more rigorous protocol, avoiding in-vitro pretreatment of the tumor cells, to confirm the previous results.
We have developed four series of compounds containing varied numbers of positively charged guanide, biguanide, phenylguanide, or naphthylguanide groups, some of which bind to CXCR4 with high affinity and could potentially interfere with CXCR4-facilitated cell metastasis 32,33. In this study, compounds with high CXCR4 affinity and low cytotoxicity were selected from more than 50 that were synthesized previously 32,33 and tested for activity in blocking CXCL12 activation of CXCR4, followed by evaluation of each compound’s ability to retard migration of breast cancer cells in an in-vitro tissue culture monolayer wound healing model. The most effective compounds (Fig. 1) were further tested in a mouse lung colonization assay to determine their capacity to reduce the number of cancer metastases.
Materials and methods
Synthesis, purification, and CXCR4 binding of the phenylguanide and naphthylguanide compounds used in this study have been described previously 32,33.
The following cell lines were used: human breast cancer cell line MDA-MB-231 (ATCC HTB-26), human T-lymphoblastic-like cell line CCRF-CEM (ATCC CCL-119), human keratinocyte cell line HaCaT 34, canine thymocyte cell line Cf2Th-CXCR4 35, and canine thymocyte cell line Cf2Th-CCR5 36. The MDA-MB-231 cells were from primary cell cultures initiated from tumor bearing SCID mice. These were passaged a maximum of six times in tissue culture flasks before intravenous injection into mice. MDA-MB-231 cells have been shown to express CXCR4 and respond to its ligand CXCL12 37–40. After the experiments described here were completed, cells were sent to ATCC for genotyping and their identity as MDA-MB-231 was confirmed.
Matrigel invasion assay
The manufacturer’s guidelines were followed for use of the BD BioCoat Matrigel Invasion Chamber (BD Biosciences, Franklin Lakes, New Jersey, USA). Briefly, 2.5×104 cells/ml MDA-MB-231 cells in Dulbecco’s modified Eagle medium (DMEM): F12 media plus 10% fetal calf serum (FCS) in the presence or absence of compound were placed in the top chamber, and DMEM: F12 plus 10% FCS, 0.4 μg CXCL12 were placed in the bottom chamber. Chambers were incubated at 37°C in the presence of 5% CO2 for 22–24 h and then unmigrated cells were removed from the top chamber by scraping the top of the membrane with a Q-tip. The lower side of the chamber membrane was fixed with methanol, stained with acid hematoxylin, and the number of migrated cells was enumerated under a microscope.
Cell viability assay
All compounds were tested for cell toxicity using the CellTiter 96AQueous Non-Radioactive Cell Proliferation Assay ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS]) (Promega Corporation, Madison, Wisconsin, USA). Nine concentrations spanning up to six orders of magnitude were used for each test compound, with the highest concentration limited by solubility or availability in a few cases. Each concentration was performed in six replicates. Cells (1×105 MDA-MB-231 or HaCat) were incubated with compound for 48–72 h. MTS was added and incubated for 1–5 h before absorbance readings were taken at 490 nm.
CXCR4 expression using flow cytometry
All cell lines were harvested and resuspended to approx. 2×105 cells/ml in PBS (0.137 mol/l NaCl, 0.016 mol/l phosphate, pH 7.2). T140-fluorescein 32 was added to the cells at a final concentration of 75 μmol/l. The mixture was incubated for 30 min on ice. Fluorescence data were collected on a FACSCalibur Flow Cytometer (BD Biosciences) and plotted as histograms of fluorescence intensity versus number of events.
Calcium flux signaling assay
The response of CCRF-CEM cells to CXCL12 was analyzed using Fluo-4 Calcium Indicators (Molecular Probes, Eugene, Oregon, USA) on the Flexstation II Scanning Fluorometer (Molecular Devices, Sunnyvale, California, USA). Cells were loaded with the Fluo-4 intracellular calcium binding dye for 1 h. After determining the optimum amount of CXCL12 to use with a standard curve for each experiment, three compound concentrations were added to separate sets of CCRF-CEM cells in duplicate wells with the Fluo-4 dye and allowed to incubate for 1 h. This was followed by the addition of CXCL12 in the Flexstation II to all the wells while monitoring the fluorescence at 525 nm.
Tissue culture wound healing migration assay
MDA-MB-231 cells were grown to confluency in DMEM: F12 (50 : 50) medium supplemented with 10% FCS. The medium was removed and a straight line of cells was detached from the monolayer using a pipette tip. Fresh media with or without compound were added to each dish. Measurements of the width of the wound (cleared zone) were taken at the time of the addition of compound and then twice daily until the ‘wound’ closed or 4–5 days later.
Alexidine dihydrochloride (100 μl of 17 μmol/l) in Tyrode’s calcium magnesium-free (CMF) saline solution was injected intravenously into seven SCID mice on days 1, 2, 5, 9, and 12. Heart punctures were carried out under anesthesia to recover blood from the mice before euthanization on day 28. The sera were submitted to the Montana Veterinary Diagnostic Lab for analysis.
Spermidine trisphenylguanide, spermidine bis-2-napthylguanide, or spermine tris-2-napthylguanide
Groups of three Balb/c mice were injected intravenously with 100 μl of 300 μmol/l spermidine trisphenylguanide, 100 μl of 100 μmol/l spermidine bis-2-naphthylguanide, or 100 μl of 100 μmol/l spermine tris-2-naphthylguanide in Tyrode’s CMF saline solution. The compound was injected on days 1, 2, and 5. On day 6, heart punctures were carried out under anesthesia to recover blood from the mice before euthanization. Sera were submitted to the Montana Veterinary Diagnostic Lab for analysis.
Lung colony metastasis assays
Female SCID mice were injected intravenously into the tail vein with 1.4×106 monodispersed MDA-MB-231 cells in 100 μl of Tyrode’s CMF saline solution. Alexidine dihydrochloride in CMF saline solution (100 μl of 17 μmol/l) was injected intravenously 24 h before the cell injection and again immediately after injection with cells. Follow-up intravenous injections of compound were administered on days 5, 9, and 12 after injection of the tumor cells. Thirty days after tumor cell injection, the mice were euthanized and lungs were harvested. Lung sets were stained with Bouin’s fixative 41 and the metastatic loads were quantified.
Spermidine and spermine derivatives
Female SCID mice were injected intravenously into the tail vein with 1×105 monodispersed MDA-MB-231 cells in 100 μl of Tyrode’s CMF saline solution. Injections of 100 μl spermidine trisphenylguanide (50, 200, or 300 μmol/l), spermidine bis-2-napthylguanide (100 μmol/l), spermine tris-2-napthylguanide (100 μmol/l), or CMF saline solution only were administered intravenously 24 h before injection with tumor cells and again immediately after injection and on day 5 after injection with cells. Thirty days after injection with tumor cells, the mice were euthanized and the lungs were removed. The lungs were stained with Bouin’s fixative and individual lung colonies were counted to determine the relative numbers of cells that extravasated out of the circulation and proliferated in the lungs.
Cellular expression of CXCR4
Flow cytometry was performed on the cell line MDA-MB-231 to verify that it expressed CXCR4 on the cell surface. A cell line that is known to overexpress the CXCR4 receptor, Cf2Th-CXCR4, was used to set the parameters for positive CXCR4 expression (Fig. 2). Cf2Th-CCR5 cells, which do not express CXCR4, were used to set parameters for negative cell populations (Fig. 2). The MDA-MB-231 human breast cancer cell line used in our studies is known to express CXCR4 on the cell surface 6 and this was confirmed in our flow cytometry assay (Fig. 2). As expected, the expression level was lower than that of the genetically manipulated Cf2Th-CXCR4 cell line.
Matrigel invasion assay
The BD Matrigel Assay was used to confirm that MDA-MB-231 cells migrated in response to exposure to CXCL12. Figure 3 shows that the addition of CXCL12 to the bottom well induced migration of the MDA-MB-231 cells through the permeable membrane. Figure 3a shows the statistical results and Fig. 3b shows images of the stained matrix. This experiment showed that MDA-MB-231 cells migrate as expected in response to CXCL12 and could therefore be used to evaluate the efficacy of compounds in inhibiting cellular migration.
Binding to CXCR4
More than 50 guanide, biguanide, phenylguanide, and naphthylguanide derivatives have been synthesized in our lab and tested for binding to CXCR4 32,33. The IC50 values for the compounds that had the highest affinity to CXCR4 are listed in Table 1.
Of all the compounds tested initially for CXCR4 affinity 32, the compounds with the highest CXCR4 affinity were spermidine bisphenylguanide and trisphenylguanide. In a later study, two naphthylguanide compounds were found to have even higher affinity 33. These compounds have IC50 values of 0.06–0.2 μmol/l (Table 1). None of these guanide derivatives bound to CXCR4 with as high an affinity as the peptide T140, which binds with a Kd of 0.43 nmol/l 42. However, T140 is not a viable therapeutic because of the fact that it is rapidly degraded in vivo43; thus, there was significant value in further testing the three synthetic compounds. Although the clearance rates of the compounds are unknown, it is reasonable to hypothesize that they would not be degraded as rapidly as the T140 peptide.
The cytotoxicity of each of these compounds was measured on the human breast cancer cell line MDA-MB-231 (Table 1) using the MTS assay with six replicates per concentration. The concentrations listed are the CC50 values, which were defined as the concentrations that yielded 50% cell death.
Two of the compounds with the highest affinity (low IC50) for CXCR4 had very low cytotoxicity, requiring greater than 500 μmol/l to kill 50% of the MDA-MB-231 cells in the assay (CC50).
Spermidine trisphenylguanide was further evaluated for toxicity on the human keratinocyte cell line, HaCaT (Table 1). The results showed that the CC50 values were similar for the two cell lines.
Calcium flux assay
To determine whether the binding of our compounds to CXCR4 was antagonistic or agonistic, the CXCL12-induced calcium flux response was evaluated.
CCRF-CEM cells endogenously express CXCR4, which was verified through flow cytometry (data not shown). The initial experiment for each assay was to determine the optimum amount of CXCL12 ligand to use. The concentration of CXCL12 that resulted in the largest intracellular calcium flux was then halved to ensure that saturation of receptors was not occurring and this concentration was then used in the experiments with the guanide compounds. The data in Fig. 4 show that the CXCR4-binding peptide T140 and the compounds spermidine bisphenylguanide and trisphenylguanide inhibited the calcium flux response in a dose-dependent manner. Therefore, these compounds were considered to be antagonists of the CXCR4 receptor. Spermine (underivatized), which did not show binding to CXCR4 in the CXCR4 inhibition assay, did not affect the intracellular calcium flux (Fig. 4).
Tissue culture monolayer wound healing assay
The guanide, biguanide, and phenylguanide compounds synthesized in a previous study 32 were screened in a tissue culture wound assay to determine whether they retarded cell migration into a cleared area. Although this assay is only semiquantitative, it was highly valuable in identifying compounds of potential interest. Two compounds that bound to CXCR4 with high affinity showed retardation of MDA-MB-231 wound recovery in this assay: Spermidine bisphenylguanide inhibited migration at 30 μmol/l and spermidine trisphenylguanide slowed migration at 30 μmol/l, but required 100 μmol/l to achieve complete inhibition of migration (Fig. 5). Neither of the starting amines showed any retardation of wound recovery at 100 μmol/l (not shown).
In-vivo mouse lung colony metastasis model
A lung colonization assay was performed to examine whether blocking the CXCR4 receptor with our guanide compounds would prevent the cells from extravasating and proliferating at sites of high CXCL12 expression, such as the lungs. Although this assay does not measure some initial events in spontaneous metastasis, lung colonization will be referred to as metastasis throughout this section. A preliminary acute toxicity study was carried out using alexidine, a biguanide compound that has already been reported to have anticancer properties 28, and spermidine trisphenylguanide. Serum from mice injected with 17 μmol/l alexidine or 300 μmol/l spermidine trisphenylguanide was evaluated for heart (creatinine kinase), kidney (blood urea nitrogen, serum creatinine), and liver toxicity (alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, bilirubin). None of the tests showed any indication of in-vivo toxicity when compared with untreated controls (data not shown).
Our lung colonization assay carried out using alexidine yielded a reduction in tumor metastasis similar to that reported previously by Yip et al.31, indicating that this mouse model was effective for evaluation of the effectiveness of guanide compounds in vivo.
Intravenous injections of spermidine trisphenylguanide (100 μl of 50 μmol/l or 200 μmol/l) reduced the number of lung metastases, but with P-values of only 0.34 and 0.3, respectively (Fig. 6a). When the dose was further increased to 100 μl of 300 μmol/l spermidine trisphenylguanide, the results showed a clearly significant reduction in the number of lung colonies (P=0.02) (Fig. 6b). A dose response was apparent for spermidine trisphenylguanide lung colony inhibition (Fig. 6a and b).
As T140 contains a naphthylalanine residue, it was hypothesized that substituting naphthylguanide groups in place of phenylguanide groups on similar backbone chains might result in compounds that bound better to the CXCR4 receptor. Accordingly, two spermidine naphthylguanide derivatives and seven spermine naphthylguanide derivatives were synthesized, and then tested for binding affinity to CXCR4 and in-vitro cytotoxicity on MDA-MB-231 cells, as described above 33. Two compounds with high affinity for CXCR4 were chosen for further study: spermidine bis-2-napthylguanide (IC50 60 nmol/l) and spermine tris-2-napthylguanide (IC50 90 nmol/l).
Because of the higher in-vitro cell cytotoxicity of the naphthylguanide-substituted compounds compared with the phenylguanide-substituted ones, they were initially evaluated for in-vivo toxicity in mice under the same experimental conditions described for spermidine trisphenylguanide. Neither 100 μl of 100 μmol/l spermidine bis-2-napthylguanide nor 100 μl of 100 μmol/l spermine tris-2-napthylguanide showed any in-vivo toxicity (heart, kidney, or liver) in our study.
These two compounds, spermidine bis-2-naphthylguanide and spermine tris-2-naphthylguanide, were then used to treat SCID mice injected intravenously with MDA-MB-231 cells to observe their effects on lung colony formation using the same protocol as that used for spermidine trisphenylguanide. The number of lung colonies was slightly reduced for spermidine bis-2-naphthylguanide (P=0.1) and significantly reduced for spermine tris-2-naphthylguanide (P=0.04) (Fig. 6c).
No extrapulmonary tumor colonies were found in any of our lung colony experiments.
We have evaluated three novel guanide CXCR4-binding compounds for their effectiveness in reducing lung colony formation by a breast cancer cell line that naturally overexpresses CXCR4. Successful candidate compounds could ultimately go onto clinical trials for breast cancer patients whose tumors overexpress CXCR4. We would not expect tumors that do not overexpress CXCR4 to be affected by these compounds. Although there are many types of neoplasms that overexpress the CXCR4 receptor, our study focused only on breast cancer metastasis to the lungs.
Initially, the most promising compound in our study was spermidine trisphenylguanide. It bound to CXCR4 with an IC50 value of 0.2 μmol/l and a CC50 of 500 μmol/l. This yields a selectivity index value (CC50/IC50) of 2500, making spermidine trisphenylguanide a promising lead for development of agents to inhibit breast cancer metastasis. The calcium flux experiments showed that spermidine trisphenylguanide was an antagonist of CXCR4 signaling and would therefore block CXCL12-induced cellular metastasis, proliferation, or vascularization.
Spermidine trisphenylguanide could inhibit in-vitro cell migration when the compound was added to a confluent layer of MDA-MB-231 cells that had a zone (wound) cleared in the monolayer, although at a 10-fold higher concentration than that required to inhibit T140 binding or CXCL12 activation of CXCR4. In the in-vivo lung colony assay, spermidine trisphenylguanide was found to be effective in reducing lung tumor colony formation at 300 μmol/l, which corresponds to 30 μmol/l when diluted into the blood volume of a mouse. The pharmacokinetics are currently unknown, but most likely the effective concentration in vivo was lower still.
Spermidine trisphenylguanide showed a clear dose response in the lung colony assay, with the 300 μmol/l (corresponding to ∼0.75 mg/kg) concentration being most effective. This concentration is still considerably lower than the CC50; thus, higher doses may be even more effective and still well tolerated.
Two additional compounds synthesized later, spermidine bis-2-napthylguanide and spermine tris-2-napthylguanide, had higher affinities for CXCR4, but were more cytotoxic than spermidine trisphenylguanide, resulting in selectivity index values of 1230 and 160, respectively. Nevertheless, initial in-vivo toxicity tests did not show any elevated serum enzymes in the mice when they were injected at 100 μl of 100 μmol/l. Therefore, they are still good candidates for further evaluation at higher concentrations. The promising in-vivo reduction in the number of lung metastases observed using concentrations of 100 μmol/l (spermidine bis-2-napthylguanide, P=0.1, and spermine tris-2-napthylguanide, P=0.04) showed that these compounds may be even more effective as CXCR4 inhibitors than spermidine trisphenylguanide, which required higher concentrations (300 μmol/l) to achieve a statistically significant reduction in the number of lung metastases (P=0.02). Further experiments increasing the concentration of these compounds in mice are warranted. Synthesis of additional compounds by changing the number and position of the hydrophobic napthylguanide groups may allow optimization of the CXCR4 affinity while minimizing toxicity.
The utility of CXCR4 inhibitors in long-term therapy to prevent CXCR4-facilitated metastasis in cancer patients is still being debated. The side effects from long-term blocking of CXCR4 may include ineffective wound healing, or mobilization of stem cells from the bone marrow to the blood, as observed in the AMD3100 trials 44, as well as cytotoxicity. Therefore, these compounds may be better suited for short-term administration immediately before and after surgery to prevent cellular metastasis when additional cells are released into the circulation.
The authors thank Lilya Kirpotina for help with designing and carrying out the calcium flux assays and the Flow Cytometry Facility at Montana State University for use of their instruments. This work was supported by Novaflux Biosciences Inc.
Conflicts of interest
There are no conflicts of interest.
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