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. 2009 Sep;11(9):901–909. doi: 10.1593/neo.09594

Synthetic Galectin-3 Inhibitor Increases Metastatic Cancer Cell Sensitivity to Taxol-Induced Apoptosis In Vitro and In Vivo1

Vladislav V Glinsky *,†, Galina Kiriakova ‡, Olga V Glinskii *,§, Valeri V Mossine , Thomas P Mawhinney , James R Turk #, Anna B Glinskii **,††, Virginia H Huxley §, Janet E Price ‡, Gennadi V Glinsky **,††
PMCID: PMC2735801  PMID: 19724684

Abstract

At present, there is no efficient curative therapy for cancer patients with advanced metastatic disease. Targeting of antiapoptotic molecules acting on the mitochondrial apoptosis pathway could potentially augment antimetastatic effect of cytotoxic drugs. Similarly to Bcl-2 family members, β-galactoside-binding lectin galectin-3 protects cancer cells from apoptosis induced by cytotoxic drugs through the mitochondrial pathway. In this study, we tested the hypothesis that inhibiting galectin-3 antiapoptotic function using a synthetic low-molecular weight carbohydrate-based compound lactulosyl-l-leucine (Lac-l-Leu) will augment apoptosis induced in human cancer cells by paclitaxel and increase its efficacy against established metastases. Treatment with synthetic glycoamine Lac-l-Leu alone reduced the number of established MDA-MB-435Lung2 pulmonary metastases 5.5-fold (P = .032) but did not significantly affect the incidence of metastasis. Treatment with paclitaxel alone (10 mg/kg three times with 3-day intervals) had no significant effect on the incidence or on the number of MDA-MB-435Lung2 metastases. Treatment with Lac-l-Leu/paclitaxel combination decreased both the number (P = .02) and the incidence (P = .001) of pulmonary metastases, causing a five-fold increase in the number of metastasis-free animals from 14% in the control group to 70% in the combination therapy group. The median number of lung metastases dropped to 0 in the combination therapy group compared with 11 in the control (P = .02). Synergistic inhibition of clonogenic survival and induction of apoptosis in metastatic cells by Lac-l-Leu/paclitaxel combination was functionally linked with an increase in mitochondrial damage and was sufficient for the antimetastatic activity that caused a reversal and eradication of advanced metastatic disease in 56% of experimental animals.

Introduction

Developing new approaches toward augmenting the efficacy of chemotherapy on advanced metastatic malignant disease is an important goal of modern cancer research. Most currently used cytotoxic drugs act by inducing neoplastic cell apoptosis through a mitochondrial pathway, which is regulated largely by the Bcl-2 family proteins (reviewed in Pommier et al. [1]). Deregulation of the Bcl-2 family in cancer cells, leading to an imbalance in a relative ratio of proapoptotic (Bax, Bak, Noxa, PUMA) to antiapoptotic (Bcl-2, Bcl-xl) members, modifies mitochondria permeabilization, determines a threshold for apoptosis induction, and contributes to the chemoresistance of malignant cells (see Pommier et al. [1] and Reed [2] for review). Thus, Bcl-2 and Bcl-2-related proteins in tumor cells have been targeted using various strategies aiming at inducing apoptosis or enhancing an apoptotic response to chemotherapy [2–5].

In the past several years, a compelling body of experimental evidence has emerged suggesting that, in addition to the Bcl-2 family proteins, a member of the galectin family, galectin-3, is also an important regulator of the mitochondrial apoptosis pathway [6,7]. This β-galactoside-binding protein shares with Bcl-2 the NWGR motif [6], which is critical for the Bcl-2 antiapoptotic activity and conserved within a BH1 domain of the Bcl-2 family [8]. Galectin-3 was shown specifically to protect cancer cells from apoptosis induced by various stimuli including serum withdrawal, nitric oxide, and several cytotoxic drugs [6,7]. On cisplatinor staurosporine-induced apoptosis, galectin-3 translocates to the perinuclear membrane and protects neoplastic cells from mitochondrial damage and cytochrome c release [7]. These observations suggest that galectin-3 protects tumor cells from apoptosis induced by cytotoxic drugs by functioning on major apoptosis execution pathways. Indeed, recent results from the group of Dr. Raz unambiguously demonstrate that galectin-3 expression regulates the apoptotic response of prostate cancer cells to chemotherapy through the mitochondrial apoptosis pathway [9]. Therefore, one can reasonably expect that blocking galectin-3 antiapoptotic function could augment the cytotoxic effect of chemotherapeutic agents on cancer cells.

Importantly, galectins could be targeted efficiently by readily available and nontoxic low-molecular weight carbohydrate-based compounds [10,11]. In this study, we investigated whether lactulosyl-l-leucine (Lac-l-Leu), a synthetic low-molecular weight carbohydrate-based galectin-3 inhibitor, would increase susceptibility to apoptosis of the human metastatic cancer cell MDA-MB-435 induced by Taxol (paclitaxel), a member of the taxane cytotoxic drug family becoming increasingly important in the therapy for various cancers. Here, we report that synthetic glycoamine Lac-l-Leu synergizes with paclitaxel to inhibit clonogenic survival and induce apoptosis in MDA-MB-435 cells to the extent sufficient to reduce paclitaxel IC50 (a concentration of the compound causing 50% inhibition) in vitro seven-fold (from 1.4 to 0.2 nM). In vivo, the Lac-l-Leu/paclitaxel combination therapy caused eradication of the establishedmetastatic disease in 56% animals and significant (>60%) inhibition of the incidence of established spontaneous metastasis in the nude mouse model. To our knowledge, this is the first study in which the reversal of the established metastatic disease using carbohydrate-based combination therapy has been documented.

Materials and Methods

Cell Lines and Culturing Conditions

The MDA-MB-435 cell line was originally isolated from a pleural effusion of a woman with metastatic breast adenocarcinoma [12]. The MDA-MB-435Lung2 variant was selected in vivo for its high potential to develop spontaneous pulmonary metastasis from mammary fat pad (MFP) tumors [13]. However, the identity of MDA-MB-435 cells as breast carcinoma has recently been challenged [14]. Nevertheless, MDA-MB-435 cells and their derivatives remain one of the most reliable in vivo models of spontaneous human cancer metastasis. Thus, to avoid further controversy, we will refer to this cell line in our study as to human metastatic cancer cells. MDA-MB-435 and MDA-MB-435Lung2 cells were maintained in culture using minimum essential medium supplemented with 5% fetal bovine serum, sodium pyruvate, nonessential amino acids, l-glutamine, and two-fold vitamin solution or RPMI-1640 medium supplemented with 2 mM l-glutamine, 100 µg/ml gentamicin, and 10% fetal bovine serum. The cultures were maintained on plastic in 5% CO2/95% air at 37°C in a humidified incubator. All cultures were free of mycoplasma and the following murine viruses: reovirus type 3, pneumonia virus, K virus, Theiler encephalitis virus, Sendai virus, minute virus, mouse adenovirus, mouse hepatitis virus, lymphocytic choriomeningitis virus, ectromelia virus, and lactate dehydrogenase virus (all assayed by MA Bioproducts, Walkersville, MD).

In Vitro Clonogenic Survival and Growth Assay

MDA-MB-435 human cancer cells grown as monolayer cultures were harvested using a standard trypsinization procedure and were pipetted to produce a single cell suspension. Live cell counts were obtained by Trypan blue dye exclusion assay. Cells were plated at a low density (200 viable cells per well) in quadruplicate in 24-well culture plates without (control samples) or in the presence of the compounds tested. Seven days later, the cells were fixed with 2% formaldehyde in PBS and stained with hematoxylin, and colonies of more than 20 cells were scored. On the basis of these experiments, the in vitro ICmin (a minimal concentration of the compound causing a statistically significant inhibitory effect) and IC50 were determined.

Apoptosis Induction Experiments

MDA-MB-435 human cancer cells were plated as described in clonogenic survival experiments without (untreated control) or in the presence of ICmin of paclitaxel and Lac-l-Leu applied as single agents or in combination. Identification of apoptotic cells was performed 24 hours after plating using the terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL) method. The In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Indianapolis, IN) that uses fluorescein-dUTP and an antifluorescein antibody conjugated to a peroxidase reporter molecule as a detection reagent was used. The results shown are representative of at least two separate experiments.

Monitoring Changes in Mitochondrial Membrane Potential

MDA-MB-435 cells were plated as for the clonogenic survival and growth assay and treated with low (ICmin) concentrations of paclitaxel, Lac-l-Leu, and their combination. After 24 hours, a 5-µg/ml solution of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) in completemediumwas added to the cultures for 10 minutes. Next, the cultures were rinsed with fresh cell culture media, and two-color ratiometric confocal microscopy was performed using a confocal microscopy system based on the Olympus IX81 inverted microscope equipped with JC-1 filter cube (Chroma Technology, Rockingham, VT) to monitor changes in mitochondrial membrane potential. At least 50 cells were observed for each experimental setting in each well.

Western Blot Analysis

For the analysis of cytochrome c release from mitochondria and caspase-3 cleavage, MDA-MB-435 cells were plated in multiple T75 flasks at the 5 x 106-cell per flask density. Next day, the cells were treated with increasing paclitaxel concentrations (0, 2.5, and 5.0 nM) with or without 500 µM of Lac-l-Leu. After 48 hours, the cells were harvested using a standard trypsinization procedure, and cytosolic fractions were obtained using a MITOISO2 mitochondria isolation kit (Sigma, St Louis, MO). Protein concentrations were determined using protein assay reagent (Bio-Rad, Hercules, CA), and 30 µg of cytosolic proteins from each sample was resolved on a 4% to 12% Nu Page Bis Tris gel (Invitrogen, Carlsbad, CA). Proteins were transferred onto a nitrocellulose membrane (Invitrogen). After blocking the membranes with 5% nonfat milk, the rabbit polyclonal antibodies against cytochrome c (no. 4272; Cell Signaling) and against caspase-3 (no. 9662; Cell Signaling, Danvers, MA), which recognize both the full-length caspase-3 (35 kDa) and the cleaved caspase-3 fragments (17 kDa), were used to analyze cytochrome c release and caspase-3 cleavage, respectively. Bound antibodies were visualized using horseradish peroxidase-conjugated secondary goat antirabbit antibody (no. 7074; Cell Signaling) and enhanced chemical luminescence system (GE Healthcare, Piscataway, NJ). Equal loading and transfer was controlled using Ponceau S staining and anti-β-actin loading control (Abcam, Cambridge, MA).

In Vivo Studies Using MDA-MB-435Lung2 Metastasis Model

A nude mouse model of spontaneous human cancer lung metastasis [12] after MFP implantation of MDA-MB-435Lung2 cells was used for evaluating the antimetastatic activity of Lac-l-Leu and paclitaxel administered alone or in combination. The care and use of the animals were in accordance with the University of Texas MD Anderson Cancer Center's Institutional Animal Care and Use Committee-approved protocol. Four- to five-week-old athymic NCr nu/nu female mice were purchased from the Animal Production Area, NCI-Frederick Cancer Research Facility (Frederick, MD). Mice were anesthetized with Metofane (PitmanMoore, Inc,Washington, NJ), and a 5-mm incision was made in the skin over the lateral thorax. The MFP was exposed, and 2 x 106 cells in a volume of 0.1 ml of PBS were injected into a fat pad. The incision was closed with wound clips. The growth of the tumors was monitored by weekly examination, and growth rates were determined as the increase in mean tumor diameter, calculated from caliper measurements of two orthogonal diameters.

Sixty-four mice were untreated until the mean tumor diameter reached 8 mm, at which time lung micrometastases are established in more than 80% of animals. At this time, daily intraperitoneal treatment began with either 0.2 ml of PBS (43 mice) or 0.2 ml of glycoamines in PBS (21 mice). The primary tumors were resected when the mean tumor diameter was 10 mm, and the mice were treated for a further 5 weeks. The second arm of the treatment with either paclitaxel (10 mg/kg; 32 mice) or cremophor vehicle (21 mice) by intravenous injection commenced on day 14 after tumor removal and was repeated twice at 3-day intervals. Mice were killed at the end of the treatment schedule, or when moribund, and examined for lung metastases both macroscopically and microscopically. The lungs and tumors were fixed in 10% buffered formalin, and paraffin-embedded sections stained with hematoxylin and eosin (H&E) were examined.

Glycoamine solutions were prepared daily by suspension of pre-weighed aliquots in sterile PBS to a final concentration of 10 µM/0.1 ml. The solutions were filtered through a 0.2-µm syringe filter before use. Paclitaxel was dissolved in Cremophor EL/ethanol (1:1 vol/vol) at the concentration of 6 mg/ml and then diluted with PBS to a final concentration of 1.25 mg/ml. Control vehicle was the same Cremophor EL/ethanol preparation diluted 1:4.8 with PBS. Paclitaxel and Cremophor EL were purchased from Sigma Chemical, Co. The drug solution was prepared on each day of injection.

Statistical Analysis, Data Calculation, and Presentation

The results of clonogenic survival and apoptosis induction experiments are presented as mean ± 1 SD. A 2-tailed paired Student's t test was used to evaluate the statistical significance of the difference between means. Statistical analysis of the incidence of lung metastasis was performed using Fisher exact test. The significance of differences in the numbers of lung metastasis was evaluated using the Mann-Whitney rank sum test.

Results and Discussion

Lac-l-Leu and Paclitaxel Cause a Synergistic Effect on Metastatic Cancer Cell Clonogenic Survival

Previously, we demonstrated that synthetic glycoamines, including Lac-l-Leu, inhibit clonogenic survival and growth of metastatic melanoma [15], breast carcinoma [11,15,16], prostate carcinoma [16], and hemangiosarcoma [17] cells by interfering with β-galactoside-mediated cell-to-cell and cell-to-matrix interactions [10,15,17]. Thus, in this study, we have used a clonogenic survival assay to investigate whether Lac-l-Leu and paclitaxel would act synergistically on MDA-MB-435 human metastatic cancer cells in vitro. First, we assessed an in vitro effect of various Lac-l-Leu concentrations on clonogenic survival of MDA-MB-435 cells. As expected, Lac-l-Leu caused dose-dependent inhibition of the MDA-MB-435 cell clonogenic survival and growth (Figure 1A) with maximal effect (complete inhibition) occurring at ∼0.63 mM. On the basis of these experiments, we also determined the in vitro ICmin and IC50 of Lac-l-Leu. In our experiments, the in vitro ICmin of Lac-l-Leu was ∼100 µM, which reproducibly caused 13% to 20% inhibition of the MDA-MB-435 cell clonogenic survival, and IC50 was ∼157 µM.

Figure 1.

Figure 1

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Synergistic effect of Lac-l-Leu and paclitaxel on MDA-MB-435 cell clonogenic survival and growth in vitro. (A) Dose-dependent inhibition of MDA-MB-435 cell clonogenic survival by Lac-l-Leu. (B) Dose-dependent effect of paclitaxel alone (solid line with open circles) or in combination with ICmin of Lac-l-Leu (solid line with closed circles) on MDA-MB-435 cell clonogenic survival and growth in vitro. A projected would-be-additive-effect graph (dotted line) was generated by extrapolating the effect of Lac-l-Leu ICmin (gray area) onto the effect of paclitaxel as a single agent (solid line with open circles). Yellow area represents a difference between the actual effect Lac-l-Leu/paclitaxel combination and a projected would-be-additive-effect graph. Note a significant shift to the left of the graph representing the effect of paclitaxel in combination with ICmin of Lac-l-Leu (solid line with closed circles) compared with the would-be-additive-effect and paclitaxel-only graphs. Red area represents a fraction of surviving cells in combination therapy experiments. (C) Effect of ICmin of paclitaxel (0.4 nM) and Lac-l-Leu (100 µM) when used as single agents or in combination on MDA-MB-435 clonogenic survival.

Next, we investigated the effect of various paclitaxel concentrations (from 0 to 3.0 ng/ml) on MDA-MB-435 in vitro clonogenic survival alone or in combination with ICmin (100 µM) of Lac-l-Leu (Figure 1B). When used as a single agent, paclitaxel inhibited MDA-MB-435 cell clonogenic survival in a dose-dependent manner, causing a complete inhibition at the concentration of ∼3.5 nM (Figure 1B, solid line with open circles). From these experiments, the in vitro ICmin of paclitaxel was determined at 0.4 nM and IC50 was determined at 1.4 nM. After that, we titrated the paclitaxel effect on MDA-MB-435 cell clonogenic survival on the background of ICmin (100 µM) of Lac-l-Leu. When paclitaxel was used in combination with the ICmin of Lac-l-Leu (Figure 1B, solid line with closed circles), the effect of paclitaxel on clonogenic survival and growth of MDA-MB-435 cells was even greater.

To determine whether this enhanced inhibition of MDA-MB-435 clonogenic survival, noted after a combined application of paclitaxel and Lac-l-Leu, resulted from a simple summation of their respective effects as single agents, or whether Lac-l-Leu indeed synergized with paclitaxel, we generated a projected “would-be-additive-effect” line by extrapolating the effects of Lac-l-Leu ICmin onto the paclitaxel dose-dependent effect (Figure 1B, dotted line). The graph representing an actual combined effect of paclitaxel and Lac-l-Leu, however, was significantly shifted to the left compared with the would-be-additive-effect graph (Figure 1B, solid line with closed circles), providing evidence that Lac-l-Leu synergizes with paclitaxel when two agents act on breast carcinoma cells simultaneously. This synergism was sufficient to cause a reduction of paclitaxel IC50 seven-fold (from 1.4 nM as a single agent to 0.2 nM in combination with Lac-l-Leu). In a separate series of clonogenic survival experiments, we used Lac-l-Leu and paclitaxel at the ICmin concentrations either as single agents or as a combination. As shown in Figure 1C, paclitaxel (0.4 nM) and Lac-l-Leu (100 µM), when applied alone, caused a 12% and 13% inhibition of clonogenic growth, respectively. However, their combined application at these same concentrations resulted in a 61% inhibition of clonogenic survival (Figure 1C).

Synergistic Effect of Lac-l-Leu/Paclitaxel Combination on Metastatic Cancer Cells Occurs through the Induction of Apoptosis Associated with a Decrease in a Mitochondrial Membrane Potential

Our next question was whether the effect of Lac-l-Leu/paclitaxel combination on metastatic cancer cells is associated with an induction of apoptosis. We therefore used a TUNEL assay to investigate whether a combination of Lac-l-Leu with paclitaxel would cause a synergistic induction of apoptosis in MDA-MB-435 cells. At 24 hours after plating, apoptosis levels in cultures treated with paclitaxel (0.4 nM) or Lac-l-Leu (100 µM) applied alone were at 13.7% and 15.8%, respectively, compared with 10.2% in the untreated control (Figure 2, A–E). However, when compounds were applied in combination, they caused an induction of apoptosis in 77.5% of MDA-MB-435 cells (Figure 2, A and E).

Figure 2.

Figure 2

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Synergistic induction of apoptosis by Lac-l-Leu/paclitaxel combination is associated with mitochondrial damage. (A) Effect of ICmin of paclitaxel, Lac-l-Leu, and their combination on the induction of apoptosis in MDA-MB-435 cells compared with the untreated control. (B–E) Neither paclitaxel (C) nor Lac-l-Leu (D) alone caused significant induction of apoptosis compared with the untreated control (B) at their respective ICmin concentrations. However, when the same concentrations of paclitaxel and Lac-l-Leu were applied together, they caused a dramatic increase in apoptosis levels (E). In B to E, clones containing at least one cell exhibiting TUNEL positivity (light or dark brown staining) are enclosed into red rectangles. (F–I) Neither paclitaxel (G) nor Lac-l-Leu (H) alone significantly compromised mitochondrial membrane potential at their respective ICmin compared with the untreated control (F). However, a combination of two drugs at the same concentrations caused a dramatic drop in mitochondrial membrane potential (I) as revealed by JC-1 staining. Numerical indices show mean ± SD percent of cells exhibiting mitochondrial catastrophe manifested by a complete or an almost complete absence of J-aggregates. (J) Western blot analysis of cytochrome c release and caspase-3 cleavage. Note that Lac-l-Leu alone did not induce either cytochrome c release or caspase-3 cleavage compared with the untreated control, whereas the Lac-l-Leu/paclitaxel combination caused a significant increase in cytochrome c release at 5.0 nM paclitaxel concentration and an elevated caspase-3 cleavage at 2.5 and 5.0 nM paclitaxel concentrations compared with paclitaxel alone.

Because our original hypothesis was that Lac-l-Leu would enhance the cytotoxic effect of paclitaxel by inhibiting galectin-3 antiapoptotic function on the mitochondrial apoptosis pathway, we next investigated the effect of paclitaxel, Lac-l-Leu, and their combination on mitochondrial membrane potential (ΔΨm). Again, we plated MDA-MB-435 cells as for the clonogenic growth assay and treated them with low (ICmin) concentrations of paclitaxel, Lac-l-Leu, and their combination. Next day, we added 5 µg/ml of cell-permeable mitochondrial probe JC-1 to the cultures for 10 minutes and used two-color ratiometric confocal microscopy to monitor changes in mitochondrial membrane potential. At high mitochondrial membrane potential, JC-1 forms “J-aggregates” exhibiting a broad excitation spectrum and an emission maximum at ∼590 nm (red fluorescence). However, as ΔΨm drops, JC-1 dissociates into a green fluorescent monomeric form, thus allowing for monitoring mitochondrial changes during apoptosis.

The results of these experiments (Figure 2, F–I) revealed that, at their respective ICmin, neither paclitaxel (Figure 2G) nor Lac-l-Leu (Figure 2H) compromised cancer cell mitochondrial membrane potential significantly compared with the untreated control (Figure 2F). However, a combination of the two drugs caused a dramatic drop in ΔΨm (Figure 2I). These results were in complete agreement with the data from the apoptosis induction experiments and confirmed that apoptosis induced in MDA-MB-435 cells by Lac-l-Leu/paclitaxel combination is associated with mitochondrial damage. Indeed, as revealed by Western analysis (Figure 2J), a combined Lac-l-Leu/paclitaxel application caused an increased cytochrome c release from mitochondria and an elevated caspase-3 cleavage in MDA-MB-435 cells, indicating further that the synergistic effect of Lac-l-Leu/paclitaxel combination converges on the mitochondrial apoptosis pathway.

The Effect of Lac-l-Leu/Paclitaxel Combination on Phosphorylation and Expression of the Bcl-2 Family Proteins

Because the mitochondrial apoptosis pathway is regulated predominantly by the Bcl-2 family, we next investigated whether the effects of Lac-l-Leu and paclitaxel on the mitochondrial apoptosis pathway are associated with changes in phosphorylation and expression of major antiapoptotic and proapoptotic Bcl-2 family proteins. In these experiments, we did not register any significant changes in the levels of expression of major antiapoptotic (Bcl-2, Bcl-xl, Mcl-1) and proapoptotic multidomain (Bax) or BH3-only (Bad, Bid, PUMA) Bcl-2 family members in response to a treatment with paclitaxel, Lac-l-Leu, or their combination (Figure 3). Paclitaxel, however, did cause a significant increase in the Bcl-2 phosphorylation on Ser70 (Figure 3, A and B). This result is consistent with previous reports from several groups showing that apoptosis induced by paclitaxel in different types of cancer is associated with an increase in Bcl-2 phosphorylation, which diminishes its ability to sequestrate proapoptotic Bcl-2 family members [18,19]. Interestingly, Lac-l-Leu did not effect paclitaxel-induced Bcl-2 phosphorylation (Figure 3, A and B). It did reduce, however, the phosphorylation of the BH3-only protein Bad on Ser112 (Figure 3, C and D) in cells treated with paclitaxel. This observation is in a complete agreement with recent results from the group of Dr. Raz identifying galectin-3-induced Bad phosphorylation on Ser112 as an important mechanism of galectin-3 antiapoptotic function in cancer cells [9]. A BH3-only protein Bad promotes apoptosis by displacing a multidomain Bax from binding to Bcl-2 and Bcl-xl. The phosphorylation of Bad on Ser112 induces Bad association with 14-3-3 protein, thus precluding its interaction with Bcl-2 and Bcl-xl. The fact that galectin-3 inhibitor Lac-l-Leu causes a significant decrease in Bad Ser112 phosphorylation suggests that this could be an important mechanism, by which Lac-l-Leu inhibits galectin-3 antiapoptotic function and augments cancer cell apoptosis induced by paclitaxel.

Figure 3.

Figure 3

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Western blot analysis of the expression and phosphorylation of major antiapoptotic (A and B) and proapoptotic (C and D) Bcl-2 family members in cells treated with Lac-l-Leu, Taxol, or their combination. Treatment with taxol caused a significant increase in Bcl-2 phosphorylation on Ser70, which was not affected by Lac-l-Leu (A and B). However, Lac-l-Leu inhibited the phosphorylation of Bad on Ser112 in cells treated with taxol (C and D), suggesting that this could be a major mechanism by which Lac-l-Leu potentiates apoptosis induced by taxol. B and D: bars show means ± SE of two independent experiments.

In Vivo Effect of Lac-l-Leu/Paclitaxel Combination on Established Metastatic Disease

Because our in vitro experiments showed that Lac-l-Leu can significantly augment the paclitaxel effect on MDA-MB-435 cells, our next question was whether it would translate into an enhanced therapeutic effect on established human cancer metastasis in vivo. Previously, we demonstrated that Lac-l-Leu is an efficient inhibitor of MDA-MB-435 spontaneous lung metastasis in vivo [11]. Thus, in this study, we have used MDA-MB-435Lung2 cells, which were selected for the enhanced ability to develop spontaneous lung metastasis from MFP tumors compared with the parental cells [13]. Further, MDA-MB-435Lung2 are twice more resistant in vivo to the effect of Lac-l-Leu ([13] and this study) and only moderately responsive to paclitaxel [20]. In addition, when designing our experiments, we attempted to model a real-life clinical situation as close as possible. Thus, after transplanting tumors into MFP, we left them untreated until the mean tumor diameter reached 8 mm. Only at this time, when lung micrometastases are already established in more than 80% of animals, daily intraperitoneal injections with either PBS or Lac-l-Leu were initiated. Primary tumors were removed surgically when their mean diameter reached 10 mm, and paclitaxel was added to the regimens on day 14 after surgery as described in the Materials and Methods section.

As shown in Table 1, daily treatment with synthetic glycoamine Lac-l-Leu alone did cause a significant reduction in the number of MDA-MB-435Lung2 spontaneous lung metastasis. The median number of pulmonary metastases was reduced 5.5-fold from 11 in control mice to 2 (P = .032) in mice treated with Lac-l-Leu (Table 1). However, the incidence of metastasis in this group was not affected significantly compared with that in the control. Although a trend toward the inhibition of the incidence of pulmonary metastasis in mice treated with Lac-l-Leu alone was noted (36% of mice in Lac-l-Leu-treated group were metastasis-free compared with only 14% in the control group), this difference was not statistically significant (Table 1). Treatment with paclitaxel alone (10 mg/kg three times with 3-day intervals) did not have a significant effect on the incidence or on the number of MDA-MB-435Lung2 metastases (Table 1).

Table 1.

Inhibition of Established MDA-MB-435Lung2 Human Cancer Metastasis in Nude Mice by Lac-l-Leu/Paclitaxel Combination.

Treatment Group Incidence of Metastasis* P Median Number of Lung Metastasis† P
Control‡ 86% (19/21) 11 (0–71)
Paclitaxel 68% (15/22) .134 7 (0–200 .39
Lac-l-Leu 64% (7/11) .147 2 (0–25) .032§
Paclitaxel + Lac-l-Leu 30% (3/10) .001§ 0 (0–30) .02§
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*

Incidence of metastasis (%) and number of mice with metastases/number of mice in parentheses. The P values are the results of Fisher exact test analyses comparing each treatment group with the control group.

†

The number of macroscopic lung metastases is shown as median and range in parentheses. The P values are the results of Mann-Whitney rank sum analyses comparing each treatment group with the control group.

‡

Cremophor vehicle control. Animals were administered cremophor vehicle intravenously on the same schedule as paclitaxel.

§

Indicates statistically significant difference compared with the control.

In contrast, when paclitaxel was used in combination with Lac-l-Leu, a significant reduction in both the incidence and the numbers of spontaneous metastases was observed (Table 1). The median number of metastases was reduced from 11 in the control group to 0 (P = .02) in mice treated with Lac-l-Leu/paclitaxel combination. The incidence of metastasis in mice treated with Lac-l-Leu/paclitaxel combination was reduced 2.9-fold (P = .0013). At postmortem examination, 70% of mice treated with Lac-l-Leu/paclitaxel combination were metastasis-free compared with only 14% in the control group (Table 1).

Additional histopathologic examination did not reveal any occult metastases that had not been detected at the necropsy. Further, many of the metastatic lesions in Lac-l-Leu/paclitaxel-treated animals appeared smaller than those in other groups both macroscopically (Figure 4, A–D) and microscopically (Figure 4, E–H). However, the number of animals with metastases and the number of metastatic lesions available for observation in a combination therapy group were too small to make a statistically meaningful comparison. On the basis of Ki-67 staining (Figure 4, I–L), there was no difference between the groups in the rate of cancer cell proliferation within pulmonary lesions. However, as manifested by an increased nuclear material leakage into the cytoplasm (Figure 4L, red arrows) and enhanced morphologic features of apoptosis such as nuclear fragmentation that could be observed with H&E staining (Figure 4P, red rectangles), enhanced apoptotic responses of tumor cells to the therapy were evident in Lac-l-Leu/paclitaxel-treated mice even at this stage. Indeed, TUNEL analysis (Figure 4, Q–T) yielded significantly higher apoptotic indices in the Lac-l-Leu/paclitaxel combination therapy group (Figure 4T) compared with those in other groups.

Figure 4.

Figure 4

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Histopathologic analysis of MDA-MB-435Lung2 spontaneous pulmonary metastasis. (A–D) High-resolution scans of the H&E-stained whole lung cross sections. (E–H) Low-magnification photomicrographs of H&E-stained lung sections. Metastatic lesions appear blue and are indicated with black arrows in panels A to D; marked with T in panels E to H. (I–L) Ki-67 staining (numerical indices show percent of Ki-67-positive cells as mean ± SD from three observation fields). In panel L, red arrows point to an increased nuclear material leakage into a cytoplasm. (M–P) H&E staining. In P, enhanced morphologic features of apoptosis such as nuclear fragmentation could be observed in the areas indicated with red rectangles). (Q–T) TUNEL analysis revealed significantly higher percentage of apoptotic (brown staining) cells in Lac-l-Leu/paclitaxel combination therapy group (T) compared with other groups. Apoptotic indices shown in the bottom left corner of each photomicrograph represent mean ± SD from three observation fields. Original magnifications: E–H, x40; I–T, x400.

The results presented in this study demonstrate that the low-molecular weight synthetic carbohydrate-based galectin-3 inhibitor Lac-l-Leu can enhance dramatically the paclitaxel effect on MDAMB-435 metastatic cancer cells in vitro and in vivo. Recently, we showed that Lac-l-Leu and modified citrus pectin (MCP), yet another carbohydrate-based galectin-3 inhibitor developed by the group of Dr. Raz [21–23], sensitize hemangiosarcoma cells to apoptosis induced by doxorubicin [17]. Further, in a related study, Chauhan et al. [24] demonstrated that inhibition of galectin-3 antiapoptotic function by MCP (GCS-100) was sufficient to reverse multiple myeloma cell resistance to bortezomib and enhance their response to apoptosis induced by dexamethasone in vitro.

Originally, both Lac-l-Leu and MCP were developed as potential antimetastatic drugs because of their ability to inhibit metastasis-associated tumor cell adhesive interactions mediated by galectin-3 [11,16,21,23]. As new evidence accumulate, indicating that galectin-3 directly regulates the sensitivity of cancer cells to various chemotherapeutic agents such as cisplatin [7,9], staurosporine [7], etoposide [9], bortezomib [24], dexamethasone [24], and doxorubicin [17], it seems that these low-molecular weight carbohydrate-based galectin-3 inhibitors may also hold the potential of dramatically enhancing cancer cell sensitivity to cytotoxic drugs by suppressing galectin-3 antiapoptotic effect on the mitochondrial apoptosis pathway. Thus, adding carbohydrate-based inhibitors of β-galactoside-mediated interactions to the treatment regimens of galectin-3.expressing malignancies may represent a new paradigm of carbohydrate-based combination therapy for cancer metastasis.

Footnotes

1

This study was supported by the VA Biomedical Laboratory Research and Development Service Merit Review Program (V.V. Glinsky), American Heart Association grant 0830287N and National Institutes of Health (NIH) P50 CA103130-01 (O.V. Glinskii), NIH HL-52490 grant (J.R. Turk), Cancer Center Support grant CA16672 (University of Texas MD Anderson Cancer Center), Charitable Leadership Foundation (Clifton Park, NY), and NIH/National Cancer Institute grant 5RO1 CA89827 (G.V. Glinsky).

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