We report here that β-galactoside binding protein (βGBP), an antiproliferative cytokine which can program cancer cells to undergo apoptosis, exhibits equal therapeutic efficacy against cancer cells that display diverse mechanisms of drug resistance and against their parental cells. The mechanisms of drug resistance in the cancer cells that we have examined include overexpression of P-glycoprotein, increased efficiency of DNA repair, and altered expression and mutation in the topoisomerase I and II enzymes. We also report that βGBP exerted its effect by arresting the cells in S phase prior to the activation of programmed cell death. The uniquely similar profile of response to βGBP by these drug-resistant cells and their parental cells extends the therapeutic potential of this cytokine in the treatment of cancers and offers a promising alternative to patients whose tumors are refractory to the currently available cadre of chemotherapeutic agents.

The ability of cancer cells to develop intrinsic or acquired resistance to virtually every drug used in cancer chemotherapy is a major problem that leads to treatment failure. It is clear that multidrug resistance is not attributable to the overexpression of the ATP-binding cassette superfamily of transporters alone (1). It is recognized that the emergence of drug resistance in cancer is a result of multiple genetic alterations during tumor progression, where changes in the expression of a large number of genes (2) contribute to drug resistance by virtue of their extended normal cellular functions in transport (3), metabolism (4), and mitogenic and survival signaling (5–7). Extensive clinical literature confirms that drug resistance is associated with poor prognosis and hence the development of novel anticancer drugs or agents that can circumvent or reverse multidrug resistance is essential in combating cancer effectively. We show here that β-galactoside binding protein (βGBP), an antiproliferative cytokine which negatively regulates the cell cycle and selectively induces apoptosis in cancer cells (8–11), extends its proapoptotic efficacy to cancer cells that have developed diverse mechanisms of drug resistance. Our results suggest that βGBP has potential application in the treatment and eradication of drug-resistant cancers.

Cell Lines. Drug-resistant oral carcinoma KB-V-1 (12), breast cancer MCF-7/D40 (13), ovarian cancer 2008/CP (14), leukemia RERC (15), and their corresponding parental cells were cultured in the appropriate media. The human colorectal carcinoma HT-29 cells had been infected with a retrovirus carrying the full-length cDNA for the human multidrug resistance gene, MDR1, which encodes P-glycoprotein, and then selected for resistance to vinblastine (16). Dose-dependent studies were conducted to determine the sensitivity of each drug-resistant cell line to βGBP compared with their respective parental cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (17). In brief, cells were plated the night before in the absence of the selecting drugs, exposed to various concentrations of βGBP for 72 hours, and followed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

Cell Growth and Apoptosis. For cell replication kinetics, triplicate cultures in 5 cm Petri dishes were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 in air. DNA content for cell cycle analysis was done on cells washed twice in PBS-bovine serum albumin, examined under an inverted microscope, fixed in 70% ethanol at 4°C, washed and stained with 40 μg/mL propidium iodide. Apoptosis was evaluated by terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling assay using the Apo-BrdUrd kit (Phoenix Flow System, San Diego, CA, USA) as described previously (11). Briefly, 1 × 106 βGBP-treated or untreated cells were washed in PBS, fixed in 1% paraformaldehyde solution, washed, resuspended in ice-cold 70% ethanol, and stored at −20°C until use. For staining, samples were incubated for 60 minutes at 37°C with terminal deoxynucleotidyl transferase enzyme and FITC-dUTP. Cells were washed, resuspended in propidium iodide and RNase solution and incubated for 30 minutes at room temperature. Samples were analyzed in a FACSCalibur (Becton Dickinson, San Jose, CA) within 3 hours of staining.

Human Recombinant βGBP. The recombinant human βGBP (Hu-r-βGBP) was expressed in E. coli BL21 using the cDNA H-Gal-1 in pET21a (18). The protein was purified by immunoaffinity chromatography using the immunoglobulin G fraction of monoclonal antibody clone B2 (8).

To determine the efficacy of Hu-r-βGBP on drug resistance, we used a panel of cancer cell lines that exhibit resistance to various chemotherapeutic agents. The drug-resistant cells include MCF-7/D40, an MCF-7-derived cell line selected for resistance to doxorubicin that overexpresses P-glycoprotein as well as other factors that contribute to the multidrug resistance phenotype (13); 2008/CP, an ovarian carcinoma cell line selected for resistance to cisplatin that exhibits increased metallothionein gene expression and enhanced DNA repair as mechanisms of resistance (14); KB-V-1, a P-glycoprotein overexpressing cell line selected for resistance to vinblastine as well as being cross-resistant to several other anticancer drugs (12); HT-29/VMDR, a multidrug-resistant HT-29 colon carcinoma cell line infected with and overexpressing the full-length cDNA for P-glycoprotein (16); and RERC, a U937-derived leukemic cell line selected for sequential resistance to etoposide and camptothecin (15). These cells were treated in parallel with their parental counterparts, either with the chemotherapeutic agents that they had been selected in (Fig. 1, top) or with Hu-r-βGBP (Fig. 1, bottom). Our results show that βGBP was equally efficient against both the parental and the drug-resistant cells regardless of their mechanisms of resistance. The IC50 of βGBP for the drug-resistant cells were between 200 and 300 ng/mL, which corresponds to the inhibitory concentrations observed in previous studies with mammary cancer cells (9, 11).

Figure 1.

Circumventing drug resistance by βGBP. Wild-type cells and their drug-resistant derivatives were treated with either the chemotherapeutic drugs that they were made resistant to or with Hu-r-βGBP and the terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling assay done 72 hours post-treatment. Points, mean; bars, ± SE of triplicate experiments.

Figure 1.

Circumventing drug resistance by βGBP. Wild-type cells and their drug-resistant derivatives were treated with either the chemotherapeutic drugs that they were made resistant to or with Hu-r-βGBP and the terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling assay done 72 hours post-treatment. Points, mean; bars, ± SE of triplicate experiments.

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In previous studies, we have shown that the modalities by which βGBP exerts its anticancer effect involve inhibition of cell replication, cell cycle arrest in S phase, and consequent activation of apoptosis (9, 11, 19). We therefore assessed rate of cell growth, cell cycle population distribution, and time of initiation and evolution of programmed cell death. Results in Fig. 2A and B show that βGBP induced dose-dependent inhibition of cell replication leading to growth arrest with a similar pattern both in MCF-7 and MCF-7/D40 cells. Terminal deoxynucleotidyl transferase–mediated nick end labeling and cell cycle analysis (Fig. 2C and D) show that the onset of apoptosis in the arrested cells became apparent at day 4 after the addition of βGBP and that apoptosis increased henceforth. Quantitation of apoptotic cells shows that there was a progressive evolution of the apoptotic process as function of time and shows that no measurable amount of cell death occurred from days 1 to 3, whereas the arrested cells accumulated in S phase (Fig. 2E, and F, inset b). These results were confirmed in repeated experiments. Similar effects on the induction of apoptosis by βGBP were observed in other drug-resistant cells and their parental counterparts (Table 1). In Fig. 2, it is noteworthy that the multidrug-resistant MCF-7/D40 cells were four times more sensitive to βGBP than their parental cells as a similar effect on inhibition of growth, S phase arrest and evolution of the apoptotic process were obtained with 50 to 100 ng/mL of βGBP instead of the 200 to 400 ng/mL required for the MCF-7 cells.

Figure 2.

Effects of βGBP on cell proliferation, cell cycle, and apoptosis. A and B, growth-response to Hu-r-βGBP according to dose (ng/mL) in MCF-7: 0 (•), 100 (▴), 200 (♦), and 400 (▪); and in MCF-7/D40: 0 (•), 25 (▴), 50 (♦), and 100 (▪). Data plotted are means of triplicate experiments. SE ranged from ± 0.02 to ± 0.54. Hu-r-βGBP added 3 hours after seeding. C and D, dual parameter terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling analysis from 20,000 events. Boxed areas, apoptotic cells and their relative percentages. Hu-r-βGBP, 400 ng/mL in MCF-7 cells and 100 ng/mL in MCF-7/D40 cells, added 3 hours after seeding. Data shown are from one representative experiment out of three. E and F, time of occurrence and pattern of progression of the apoptotic process in cells treated with Hu-r-βGBP (400 ng/mL in MCF-7, 100 ng/mL in MCF-7/D40) from 3 hours after seeding (filled columns) and parallel untreated controls (open columns). Data represent means of percentages of apoptotic cells from three separate experiments. Standard error ranged from ± 0.07 to ± 3.54. Insets, cell cycle distribution of DNA content assessed by fluorescence-activated cell sorting analysis at day 3, prior to the manifestation of apoptosis: a, cycling control cells; b, βGBP-arrested cells.

Figure 2.

Effects of βGBP on cell proliferation, cell cycle, and apoptosis. A and B, growth-response to Hu-r-βGBP according to dose (ng/mL) in MCF-7: 0 (•), 100 (▴), 200 (♦), and 400 (▪); and in MCF-7/D40: 0 (•), 25 (▴), 50 (♦), and 100 (▪). Data plotted are means of triplicate experiments. SE ranged from ± 0.02 to ± 0.54. Hu-r-βGBP added 3 hours after seeding. C and D, dual parameter terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling analysis from 20,000 events. Boxed areas, apoptotic cells and their relative percentages. Hu-r-βGBP, 400 ng/mL in MCF-7 cells and 100 ng/mL in MCF-7/D40 cells, added 3 hours after seeding. Data shown are from one representative experiment out of three. E and F, time of occurrence and pattern of progression of the apoptotic process in cells treated with Hu-r-βGBP (400 ng/mL in MCF-7, 100 ng/mL in MCF-7/D40) from 3 hours after seeding (filled columns) and parallel untreated controls (open columns). Data represent means of percentages of apoptotic cells from three separate experiments. Standard error ranged from ± 0.07 to ± 3.54. Insets, cell cycle distribution of DNA content assessed by fluorescence-activated cell sorting analysis at day 3, prior to the manifestation of apoptosis: a, cycling control cells; b, βGBP-arrested cells.

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Table 1.

Effects of βGBP on cell death in wild-type and drug-resistant cancer cell lines

Cell linesMCF-7MCF-7/D4020082008/CPHT-29HT-29/VMDRU937RERC
% of Apoptotic cells* 80 78 76 82 89 80 65 75 
Cell linesMCF-7MCF-7/D4020082008/CPHT-29HT-29/VMDRU937RERC
% of Apoptotic cells* 80 78 76 82 89 80 65 75 
*

Results shown were representative of one of three independent measurements.

The effectiveness of current cancer chemotherapeutic drugs is hampered by lack of specificity, systemic toxicity, rapid drug metabolism, and the development of either intrinsic or acquired drug resistance (20). The effectiveness of drugs designed to target specific molecular lesions responsible for the deregulated growth of cancer cells is confounded and hampered by the multiplicity of lesions, which optimal agent to match to which lesion, toxicity, and resistance. In the present study, we have shown that βGBP exhibits equal therapeutic efficacy against cancer cells whether or not they have developed drug resistance. The cancer cells we have examined include those that overexpressed P-glycoprotein (MCF-7/D40, KB-V-1, and HT-29), those with increased DNA repair and metallothionein overexpression (2008/CP), and those with altered expression or mutation in the topoisomerase I (RERC) or II (MCF-7/D40 and RERC) enzymes. Significantly, the different mechanisms of drug resistance operating in these cells represent some of the most relevant causes of resistance in cancer known to date.

The basis for the ability of βGBP to equally exert its proapoptotic effect regardless of the multiple genetic and biochemical changes, which in cancer cells fosters escape from therapeutic attack, is implicit, conceivably, with the nature and function of the βGBP molecule. Unlike conventional chemotherapeutic agents and drugs designed for molecular targeting, βGBP is a physiologically occurring antiproliferative cytokine. Secreted by CD4+- and CD8+-activated T cells (10), βGBP is also endogenously released by somatic cells where it modulates cell cycle transition from S phase to G2-M (8). At cytostatic concentrations (20-400 ng/mL), the recombinant protein binds with high affinity (Kd 10−10 mol/L) to specific cell surface receptors to induce reversible S phase arrest in normal cells. By contrast, in cancer cells, cell cycle arrest is followed by programmed cell death (9, 11, 19), a pattern also observed in this study (Fig. 2).

Which molecular events regulated by βGBP are involved in S phase control in normal cells and in determining the shift from growth arrest to apoptosis in cancer cells is still under investigation. In previous work, we have shown that βGBP reduces Bcl-2 levels by shifting the Bcl-2/Bax ratio in favor of Bax (19). Recently, we have reported a correlation between βGBP-induced cell cycle arrest and deregulated transactivation of the E2F1 transcription factor as a condition that can lead cancer cells into apoptosis (9). Thus, apoptosis by βGBP could be initiated through modulation of existing apoptotic regulatory proteins and through transcription and translation-dependent changes to which normal and cancer cells respond differently. However, evidence is emerging that other pathways which regulate the cell growth/apoptosis equation can also be affected by βGBP.3

3

L. Mallucci and V. Wells, unpublished data.

It is conceivable that the exploitation of multiple proapoptotic pathways by βGBP adds to its ability to bypass drug resistance and provides a likely rationale, based on chance differences in molecular make-up, for the greater sensitivity to βGBP that drug-resistant cells can have with respect to their parental cells (Fig. 2).

Produced by activated T cells, βGBP is a naturally occurring molecule circulating in the healthy organism whose anticancer properties suggests a role in cancer surveillance and indicate a conceivably new mode by which immune cells may control malignancy. There are currently no chemotherapeutic drugs in the clinic that have the broad-spectrum of antitumor activity together with the ability to overcome multiple mechanisms of drug resistance as shown by βGBP. This suggests that βGBP offers a potentially safe and novel approach in cancer therapy which may be of particular benefit to patients who no longer respond to current chemotherapeutic treatments.

Grant support: Breast Cancer Campaign, UK (V. Wells and L. Mallucci) and the New Jersey Commission on Cancer Research (K-V. Chin).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Jun Hirabayashi for the gift of Hu-βGBP cDNA construct, Derek Davies for generous help with FACS and TUNEL analysis, and Kate Kirwan for patient and skilful artwork.

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