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Clinical Cancer Research 13, 2658-2666, May 1, 2007. doi: 10.1158/1078-0432.CCR-06-2642
© 2007 American Association for Cancer Research
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Cancer Therapy: Clinical

Bcl-2 and Glutathione Depletion Sensitizes B16 Melanoma to Combination Therapy and Eliminates Metastatic Disease

Salvador Mena1, María Benlloch1, Angel Ortega1, Julian Carretero1, Elena Obrador1, Miguel Asensi1, Ignacio Petschen2, Bob D. Brown3 and José M. Estrela1

Authors' Affiliations: 1 Department of Physiology, Faculty of Medicine and Odontology, University of Valencia, and 2 Radiotherapy Service, La Fe Hospital, Valencia, Spain; and 3 Genta, Berkeley Heights, New Jersey

Requests for reprints: José M. Estrela, Department of Physiology, Faculty of Medicine and Odontology, University of Valencia, 17 Av. Blasco Ibanez, 46010 Valencia, Spain. Phone: 34-963864646; Fax: 34-963864642; E-mail: jose.m.estrela{at}uv.es.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Advanced melanoma resists all current therapies, and metastases in the liver are particularly problematic. Prevalent resistance factors include elevated glutathione (GSH) and increased expression of bcl-2 in melanoma cells. GSH has pleiotropic effects promoting cell growth and broad resistance to therapy, whereas Bcl-2 inhibits the activation of apoptosis and contributes to elevation of GSH. This study determined the in vivo efficacy of combination therapies administered while GSH and Bcl-2 were individually and simultaneously decreased in metastatic melanoma lesions.

Experimental Design: Highly metastatic murine B16 melanoma (B16M-F10) cells have elevated levels of both GSH and Bcl-2. B16M-F10 cells were injected i.v. to establish metastatic lesions in vivo. GSH was decreased using an L-glutamine–enriched diet and administration of verapamil and acivicin, whereas Bcl-2 was reduced using oligodeoxynucleotide G3139. Paclitaxel, X-rays, tumor necrosis factor-{alpha}, and IFN-{gamma} were administered as a combination therapy.

Results: Metastatic cells were isolated from liver to confirm the depletion of GSH and Bcl-2 in vivo. Reduction of Bcl-2 and GSH, combined with partial therapies, decreased the number and volume of invasive B16M-F10 foci in liver by up to 99% (P < 0.01). The full combination of paclitaxel, X-rays, and cytokines eliminated B16M-F10 cells from liver and all other systemic disease, leading to long-term survival (>120 days) without recurrence in 90% of mice receiving the full therapy. Toxicity was manageable; the mice recovered quickly, and hematology and clinical chemistry data were representative of accepted clinical toxicities.

Conclusions: Our results suggest a new strategy to induce regression of late-stage metastatic melanoma.

The proto-oncogene bcl-2 is directly associated with cellular transformation and resistance chemotherapy (1, 2). Although aberrant expression of bcl-2 occurs in virtually all melanomas and overexpression has been associated with significantly shorter patient survival (3–5), the relationship between bcl-2 expression and resistance to therapy in vivo remains controversial. Bcl-2 homologues, alternative regulatory pathways, and other mechanisms contribute to the resistant phenotype (2). Nevertheless, reduction of Bcl-2 levels increases the activity of chemotherapy against many tumor types in vitro and in vivo (6, 7) and increases sensitivity to endothelium-induced cytotoxicity and to proapoptotic agents such as tumor necrosis factor-{alpha} (TNF-{alpha}) in vivo (8, 9).

Antisense oligodeoxynucleotides targeted against bcl-2 (bcl-2–AS) are short, single-stranded nucleic acids that mediate the degradation of bcl-2 RNA. bcl-2–AS was first introduce by Reed (1) and has been extensively evaluated in the form of G3139 (oblimersen sodium, Genta Incorporated). Systemic administration of G3139 to prostate cancer–bearing mice led to a rapid decrease of tumor size (higher when chemotherapy was simultaneously administered; ref. 10). G3139-induced tumor regression without dose-limiting toxicity was also observed in other tumors, e.g., melanoma, lymphoma, or gastric cancers (11). Synergism of the G3139 and anticancer drugs was also shown in different tumors, including human melanoma (7, 12, 13). Clinical trials showed that G3139 has an acceptable clinical safety profile and positive impact on several clinical end points; nevertheless, significant improvements are still needed (14). Its limited effect points out the importance of developing methods to improve the clinical efficacy of bcl-2 antisense therapy in general and in metastatic melanoma in particular.

Glutathione ({gamma}-glutamyl-cysteinyl-glycine; GSH) is involved in many cellular functions, including bioreductive reactions, maintenance of enzyme activity, amino acid transport, detoxification of xenobiotics, mitogenic stimulation, and regulation of DNA synthesis (15). In tumor cells, GSH controls tumor-cell proliferation by regulating protein kinase C activity and intracellular pH (16), and increased GSH promotes growth of metastatic cells (17, 18). Resistance to chemo- and radiotherapy is also frequently associated with high GSH content, and GSH depletion can restore sensitivity to radiotherapy (17, 19). GSH also protects normal cells against oxidative stress, and elevated GSH protects metastatic melanoma cells from the increased oxidative/nitrosative stress found within the hepatic microvasculature (20).

Numerous derivatives of the murine B16 melanoma (B16M) cell line are used in metastases research due to differing degrees of metastatic activity and resistance to therapies. Analysis of the bcl-2 family of genes revealed that B16M-F10 cells (high metastatic potential) overexpressed bcl-2 relative to B16M-F1 cells (low metastatic potential; ref. 21). B16M-F10 cells have higher intracellular GSH levels than B16M-F1 cells due to faster GSH synthesis and slower GSH efflux (8, 22). In addition to direct antiapoptotic functions, such as inhibition of cytochrome c release, Bcl-2 inhibits the release of GSH through the cystic fibrosis transmembrane conductance regulator [CFTR; a multidrug resistance protein 1 (MRP1)–like member of the ABC family of transport proteins], which further enhances GSH accumulation and B16M-F10 resistance to cytotoxic agents (8). Depletion of Bcl-2 has been confirmed to decrease intracellular GSH (8).

Verapamil accelerates the loss of GSH from B16M-F10 cells by activation of MRP1 (8). Acivicin blocks {gamma}-glutamyl transpeptidase ({gamma}-GT; ref. 22). {gamma}-GT is crucial for recycling L-cysteine (Cys) from the extracellular pool of GSH (22, 23). Because lower Cys levels limit de novo GSH synthesis, simultaneous treatment of tumor-bearing animals with verapamil and acivicin can deplete GSH levels in B16M-F10 cells in vivo (8).

Finally, many tumors exhibit a remarkable preference for L-glutamine (Gln) as a respiratory fuel, and B16M-F10 cells oxidize Gln to L-glutamate (Glu) at a high rate (24). Glu subsequently inhibits mitochondrial uptake of GSH, which renders metastatic cell mitochondria more susceptible to oxidative stress (9). Under in vitro conditions, Gln-adapted B16M-F10 (B16M-F10-Gln+) cells are more sensitive to TNF-{alpha}–induced oxidative stress, and this damaging effect can be further potentiated by bcl-2–AS–induced Bcl-2 depletion (9). However, this sensitizing strategy has not been tested in combination with chemotherapy and/or radiotherapy in vivo.

We have investigated whether combined depletion of Bcl-2 and GSH increases the antitumor efficacy of biotherapy, chemotherapy, and/or ionizing radiation against the B16M-F10 model. Our results show that the combination strategy leads to a complete regression of metastatic melanoma tumors.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Murine B16M-F10 (from the American Type Culture Collection) cells were cultured in DMEM (Life Technologies; pH, 7.4), supplemented with 10% FCS (Life Technologies), 10 mmol/L HEPES, 40 mmol/L NaHCO3, 100 units/mL penicillin, and 100 µg/mL streptomycin (20). Cell integrity was assessed by trypan blue exclusion and leakage of lactate dehydrogenase activity (20).

Animals and diets. Syngenic male C57BL/6J mice (9 weeks old) from Charles River Laboratories were fed ad libitum on a standard diet or an equivalent diet but glutamine (Gln)-enriched diet (GED; 15% of total dietary nitrogen from Gln; Letica; ref. 24). Both diets were isonitrogenous and isocaloric. Mice were kept on a 12-h-light/12-h-dark cycle with the room temperature maintained at 22°C. Procedures involving animals were in compliance with international laws and policies (European Economic Community Directive 86/609, OJ L 358. 1, December 12, 1987; and NIH Guide for the Care and Use of Laboratory Animals, NIH Publ. No. 85-23, 1985).

Local tumor growth. B16M-F10 cells were harvested from culture flasks by exposure to 0.02% EDTA (5 min at 37°C), washed twice in DMEM, resuspended in the same culture medium, and injected into the footpad of the right hind-limb (104 cells/20 µL). Local tumor growth was determined by measuring footpad diameter with calipers every 2 days, starting on the first day of treatment. Tumor size was calculated according to the formula: tumor diameter = (diameter of footpad with growing tumor) – (diameter of DMEM-treated contralateral footpad).

Isolation of B16M-F10 and B16M-F10-Gln+ cells growing in vivo. Anti-Met72 monoclonal antibodies and flow cytometry–coupled cell sorting were used to isolate viable B16M-F10 or Gln-adapted B16M-F10 (B16M-F10-Gln+; ref. 9) cells (from the tumors growing in the footpads or from metastases in the liver of mice fed, respectively, a standard diet or a GED; ref. 25). Anti–Met72 monoclonal antibodies, which react with a 72-kDa cell-surface protein (Met72) expressed at high density on B16M clones of high metastatic activity, were produced as previously described (26) through syngenic immunizations of C57BL/6J mice with clones of B16M-F10. Tissues containing tumor cells were obtained by surgical means. Cell dispersion was carried out in minced tissues by a sequential procedure that includes (a) trypsinization; (b) three washes in PBS; and (c) collagenase digestion (26). Then, cells were washed thrice in PBS and resuspended in DMEM, and an aliquot containing 2 x 106 cells was incubated with a predetermined excess of anti–Met72 monoclonal antibody for 1 h on ice. After three washings with PBS, cells were incubated with FITC-conjugated sheep anti-mouse immunoglobulin F(ab)2 [IgF(ab)2] (Cappel Laboratories) for 1 h on ice. After another three washing steps with PBS at 4°C, cell pellets were resuspended in 1 mL ice-cold PBS, filtered through a 44-µm pore mesh and analyzed using a MoFlo High-Performance Cell Sorter (DAKO). Fluorescent melanoma cells were separately gated for cell sorting and collected into individual tissue culture chambered slides (Nalge Nunc International Corp.). Then, the sorted tumor cells were harvested and plated in 25-cm2 polystyrene flasks (Falcon Labware) as above.

Experimental metastases. Hepatic metastases were produced by i.v. injection (portal vein) into anesthetized mice (Nembutal, 50 mg/kg i.p.) of 105 viable B16M-F10 or B16M-F10-Gln+ cells suspended in 0.2 mL DMEM. Mice, which were fed a standard diet or a GED, were cervically dislocated 10 days after tumor cell inoculation. The livers were fixed with 4% formaldehyde in PBS (pH, 7.4) for 24 h at 4°C and then paraffin embedded. Metastasis density (mean number of foci/100 mm3 of liver detected in fifteen 10 x 10-mm2 sections per liver) and metastasis volume (MV, mean % of liver volume occupied by metastasis) were determined as earlier described (18).

Analysis of Bcl-2 levels. For Western blotting, cultured cells, harvested as indicated above, or minced tissues were washed twice in ice-cold Krebs-Henseleit bicarbonate medium (pH, 7.4). Cell or tissue extracts were made by freeze-thaw cycles (cells) or homogenization (tissues) in a buffer containing 150 mmol/L NaCl, 1 mmol/L EDTA, 10 mmol/L Tris-HCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, 1 µg/mL aprotinin, and 1 µg/mL pepstatin (pH, 7.4). About 50 µg of protein (as determined by the Bradford assay) were boiled with Laemmli buffer and resolved in 12.5% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and subjected to Western blotting with anti-mouse Bcl-2 monoclonal antibody (clone YTH-10C4 from Trevigen, Inc.). Blots were developed using horseradish peroxidase–conjugated secondary antibody and enhanced chemiluminescence (ECL system, Amersham).

Bcl-2 protein was quantitated in the soluble cytosolic fraction by enzyme immunoassay (21) using a monoclonal antibody–based assay from Sigma (1 unit of Bcl-2 was defined as the amount of Bcl-2 protein in 1,000 nontransfected B16M cells).

GSH measurement. Organs, tissues, or tumor cells were obtained and treated as previously described (18, 19). GSH was measured by the glutathione S-transferase reaction (16).

Antisense bcl-2 oligodeoxynucleotides. Fully phosphorothioated 18-mer bcl-2–AS were from Genta: G3139 (human; sequence: 5'-TCTCCCAGCGTGCGCCAT-3'), G4244 (murine; sequence: 5'-TCTCCCGGCTTGCGCCAT-3', two differences from G3139 sequence), and G3622 (reversed G3139 sequence control).

Cytokines. Recombinant murine TNF-{alpha} (rmTNF-{alpha}, 2 x 107 units/mg protein) and recombinant murine IFN-{gamma} (rmIFN-{gamma}; 105 units/mg protein) were obtained from Sigma. Stock solutions (5 x 105 units rmTNF-{alpha}/mL and 25 x 104 units rmIFN-{gamma}/mL) were diluted in sterile physiologic saline solution (0.9% NaCl); adjusted to pH, 7.0; and stored at 4°C.

Irradiation procedure. Whole mice were irradiated with X-rays using a 6-keV SL75 linear accelerator from Philips. For this purpose, animals were placed in a Perspex box divided by 0.5-cm Perspex plates into small chambers of 7 x 7 x 10 cm (one mouse per chamber). Single-fraction radiotherapy, at total doses ranging from 1.0 to 6.0 Gy (below the LD50 for mice; ref. 19), was administered at a rate of 5.0 Gy/min.

Evaluation of therapy-induced in vivo toxicity. This included the following parameters: animal weight, complete blood cell count, and standard blood chemistry.

Immunohistochemical detection of metastatic cells. Anti-S100 monoclonal antibodies (DAKO) were used for immunohistochemical detection of metastatic B16 melanoma cells (21). For that purpose, the livers were fixed and paraffin embedded as described above under Experimental Metastases. Immunohistochemical analysis was applied to tissue slices (5 µm thick) following the standard methodology recommended by DAKO. Anti-rabbit IgG, horseradish peroxidase-linked antibodies (Cell Signaling Technology), were used for secondary detection.

In vivo detection of cell proliferation. 5-Bromo-2-deoxy-uridine (BrdUrd) labeling and detection in liver tissue sections (Fig. 2) were done using a standard kit from Roche, which detects BrdUrd-labeled DNA with an anti-BrdUrd antibody and then makes the antibody-labeled DNA visible with a fluorescein-labeled anti-mouse secondary antibody. Anti-BrdUrd antibody (20 mg/kg) was injected i.v. 3 h before sacrifice. The livers were fixed, paraffin embedded, and cut as described above.


Figure 2
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  		Fig. 2. Liver metastases 10 d after B16M-F10-Gln+ cell inoculation in mice treated with (A) physiologic saline and (B) G3139 + verapamil + acivicin + paclitaxel + X-rays. For each experimental condition, the following are shown: macroscopic image of the liver, immunochemical detection of metastatic cells using S100 monoclonal antibodies as markers of melanocytic tumors (brown staining), and cell proliferation detection using BrdU.

 
Statistical analysis. Data were analyzed by Student's t test.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Bcl-2 depletion in metastatic B16M-F10 cells. Antisense oligonucleotides G3139 and G4244 (human and murine, respectively, bcl-2–AS) both depleted murine Bcl-2 levels to a similar extent in B16M-F10-Gln+ cells growing in vitro (and in control B16M-F10 cells, data not shown), whereas the control oligonucleotide G3622 had no effect (Fig. 1A ). Consistent results were obtained in vivo by isolating metastatic cells from the livers (see Materials and Methods) of mice bearing B16M-F10-Gln+ tumors treated with G3139 or the G3622 control oligonucleotides. G3139 treatment (and G4244, data not shown) decreased Bcl-2 levels in the metastatic cells in vivo to ~52% of control values (Fig. 1B). No significant change in Bcl-2 protein level was detected in parenchymal hepatocytes, suggesting differential activity of G3139 and G4244 on melanoma cells versus normal liver cells (Fig. 1B). The lack of anti–Bcl-2 activity in normal tissues was confirmed in several other normal tissues isolated from the tumor-bearing mice (Fig. 1C). Consistent with our results, bcl-2 expression in normal murine tissues was unchanged after systemic administration of G3139 in mice bearing prostate tumor xenografts (10). Because both bcl-2–AS sequences evaluated, G3139 and G4244, have similar effects on Bcl-2 levels in vitro and in tumor and nontumor cells in vivo, we selected G3139 for testing.


Figure 1
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  		Fig. 1. Effect of bcl-2-AS on Bcl-2 in tumor cells and nontumor tissues. A, Western blotting with antimouse Bcl-2 monoclonal antibody in B16M-F10-Gln+ cultured cells treated in vitro with G3139, G4244, or G3622. blc-2–AS (1 µmol/L) were added to the culture medium each 24 h (starting 12 h after seeding and proteins were extracted 12 h after the third addition). To facilitate in vitro uptake of oligodeoxynucleotides, cytofectine (1.25 µg/mL; Genlantis) was used in the cell cultures as a lipophilic transfection reagent. B, Quantification of Bcl-2 by enzyme immunoassay in B16M-F10-Gln+ cells and hepatocytes isolated from metastasized livers of mice treated with physiologic saline or G3139 (10 mg/kg/d x 6 d). Columns, means of four to five different experiments; bars, SD. *, P < 0.01 comparing G3139- versus physiologic saline-treated mice. C, Western blot analysis of Bcl-2 levels in different tissues from mice bearing B16M-F10-Gln+ metastatic cell foci and treated with physiologic saline (–) or G3139 (+; as in B). (A) and (C) correspond, in each case, to a representative experiment of four similar experiments.

 
GSH depletion in metastatic B16M-F10 cells. The total GSH content of metastatic B16M-F10-Gln+ cells in vivo (isolated from livers as above) and in normal tissues after administration of saline and treatment with G3139 alone, G3139 + verapamil (which activates GSH efflux via CFTR and MRP1), and G3139 + verapamil + acivicin (where, in addition, the Cys supply for GSH synthesis is limited by inhibiting {gamma}-GT) is shown in Table 1 . GSH was significantly lower in liver, kidney, and pancreas of saline-treated tumor-bearing mice, as compared with non–tumor-bearing mice. G3139, administered alone to B16M-F10-Gln+–bearing mice, did not further deplete GSH in either tumor or normal cells. Note that significantly higher GSH levels were measured in B16M-F10-Gln+ cells taken from G3139 + verapamil–treated mice. This was due to a Cys-dependent rebound effect on GSH synthesis and can be prevented by acivicin (8).


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  		Table 1. GSH content in tumor cells and tissues from B16M-F10-Gln+–bearing mice treated with Bcl-2-AS, verapamil, and/or acivicin

 
Triple-combination therapy with G3139 + verapamil + acivicin decreased tumor cell GSH content to ~43% of control values without affecting GSH in normal tissues (Table 1). Similar results were obtained by treatment with G4244 + verapamil + acivicin (data not shown). When the inactive reversed sequence oligo-G3622 was administered with verapamil and acivicin, GSH levels were similar to those displayed in Table 1, except that this combination was unable to decrease GSH content in B16M-F10-Gln+ cells (40 ± 5 nmol/106 cells, n = 7). Moreover, as previously described (9, 24), a GED induced mitochondrial GSH (mtGSH) depletion in the metastatic cells, but not in normal cells from mice treated with physiologic saline. Indeed, in B16M-F10-Gln+–bearing mice treated with physiologic saline or G3139 + verapamil + acivicin, 8 days after tumor inoculation, mtGSH in the metastatic cells decreases to 3.1 ± 0.5 and 2.7 ± 0.5 nmol/106 cells, respectively (n = 5-6, P < 0.01; ~45% and 39% of the mtGSH levels measured in metastatic B16M-F10 cells isolated from the liver of mice fed a standard diet). We found previously that death in B16M-F10 cells was sharply activated at mtGSH levels below 30% of control values (21), which is critical because TNF-{alpha}–induced cytotoxicity is facilitated in B16M-F10 cells with low mtGSH levels (9, 25).

Elimination of metastatic tumors in vivo by combined biochemotherapy and X-rays. As shown above, our experimental approach decreased Bcl-2 (Fig. 1) and GSH (Table 1) levels in metastatic melanoma cells in vivo, but not in normal cells and, therefore, might enhance the efficacy of conventional therapies (building on the results of Benlloch et al. and Ortega et al.; refs. 9, 21). We used a combination of biochemotherapy and radiation to test this hypothesis. Thus, we designed a protocol based on three basic ideas: (a) cytotoxic chemotherapy and radiation would be administered early to cause as much damage as possible in the metastatic cell population; (b) Bcl-2 and GSH depletion would keep the damaged survivors sensitive to oxidative stress; (c) administration of biotherapy to increase oxidative stress sufficiently to kill surviving cells. X-rays and paclitaxel were selected on the basis of previous studies, including screening most chemotherapeutic drugs used against human melanoma cells, including daunorubicin, vincristine, vindesine, vinblastine, bleomycin, methotrexate, arsenate, cisplatin, carmustine, and dacarbazine (9).

Drug doses and administration sequences (see the caption in Table 2 ) were evaluated in preliminary studies to optimize each element in combination-treatment protocols. As shown in Table 2, G3139 + verapamil + acivicin treatment decreased metastatic B16M-F10-Gln+ tumor volume (MV) by ~83% as compared with saline-treated controls. Addition of paclitaxel or X-rays to the Bcl-2– and GSH-depleting protocol decreased MV by ~96% and ~99%, respectively (Table 2). Combined administration of G3139 + verapamil + acivicin + paclitaxel + X-rays decreased MV to ~0.01% of control values (Table 2), and all remaining B16M-F10-Gln+ cells were found either as small groups of ~5 to 10 cells or as single isolated cells within the liver tissue (Fig. 2 ). In the absence of any additional therapy, these small numbers of isolated cells gave rise to lethal recurrent metastatic tumors (data not shown) and short survival durations (Fig. 3 ). However, the addition of cytokines (TNF-{alpha} and IFN-{gamma}) reduced surviving tumor cells to undetectable levels in 100% of the G3139 + verapamil + acivicin + paclitaxel + X-rays–treated mice (Table 2). Most mice (90%, Fig. 3) treated with the full combination regimen were completely cleared of tumor cells, as shown by survival beyond 120 days with no detectable tumor recurrence. The timing and order of treatment (see Table 2) had significant impact on the efficacy of the curative regimen. If paclitaxel and X-rays were administered on days 6 and 7 and cytokines on days 8 and 9, the MV was never below of ~4-5% in mice receiving the full treatment (data not shown).


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  		Table 2. Effect of Bcl-2 and GSH depletion-induced sensitization to combined biochemotherapy and ionizing radiations on B16M-F10-Gln+ melanoma growth in the liver

 

Figure 3
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  		Fig. 3. Effect of treatment-induced suppression of metastatic B16M-F10-Gln+ melanoma growth on host survival. Tumor-bearing mice were treated as in Table 2. Host survival was studied in the following treatment conditions: ({circ}) physiologic saline; (bullet) G3139 + verapamil + acivicin; ({square}) G3139 + verapamil + acivicin + X-rays; ({blacksquare}) G3139 + verapamil + acivicin + X-rays + paclitaxel; ({bigtriangleup}) G3139 + verapamil + acivicin + X-rays + paclitaxel + rmTNF-{alpha} + rmIFN-{gamma}. Data are means ± SD for 20 different mice in each experimental condition.

 
Evaluation of therapy-induced systemic toxicity. Standard hematology and clinical chemistry were measured to evaluate the side effects of the treatment regimen that eliminated metastatic B16M-F10-Gln+ cells from the majority of treated mice. As shown in Table 3 , side effects included anemia, lymphopenia, severe neutropenia, and an increase of several tissue-damage–related enzyme activities in plasma, including aspartate aminotransferase, alanine aminotransferase, {gamma}-GT, alkaline phosphatase, and lactate dehydrogenase. However, in the mice cleared of tumor cells (90%, >120-day survival; Fig. 3), all hematologic and clinical chemistry measures returned to normal values measured in untreated, non–tumor-bearing mice by 40 days after treatment (Table 3). Although the side effects measured in the treated mice are indeed significant, such alterations are commonly observed and managed in melanoma patients receiving clinical therapies.


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  		Table 3. Hematology and clinical chemistry data in B16M-F10-Gln+–bearing mice treated to induce suppression of metastases

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Overexpression of bcl-2 is associated with poor outcomes in several cancers and correlates with the progression of both human and murine cancer cells to a metastatic phenotype (22, 27–29). Bcl-2 expression and/or function has been inhibited in tumor cells in vitro and in vivo using multiple, independent strategies, including small molecules, antisense G3139, non-G3139 antisense oligonucleotides, and small interfering RNAs, and all have been shown to have direct antitumor cell efficacy or to increase the efficacy of other agents (7, 11–13, 30–32). These results validate Bcl-2 as an important therapeutic target in multiple tumor types. However, the literature contains conflicting conclusions on the impact of bcl-2 expression in tumor cells. Recent results suggest that some of these inconsistencies could be due to different roles of Bcl-2 in vitro versus in vivo experimental models (33). For example, human melanoma 518A2 cells express bcl-2. When implanted into immunodeficient mice, 518A2 cells grew rapidly into lethal tumors. Cell lines derived from 518A2 with lowered Bcl-2 levels were equivalent to parental cells by several measures in vitro, including cell growth, but failed to grow in vivo or formed only small tumors and then regressed. Down-regulation of Bcl-2 has been measured in mononuclear cells and melanoma biopsies from patients treated with G3139 (6, 34). However, although phase III trials in both malignant melanoma and chronic lymphocytic leukemia patients confirm that patients receiving G3139 had more remissions and longer duration remissions than those receiving chemotherapy alone, the differences were not enough to warrant approval by the U.S. Food and Drug Administration (30).4

Different reports indicated that GSH depletion may have therapeutic use in sensitizing bcl-2–overexpressing cells to cell death (ref. 21 and references therein). Recently, we reported that in vitro treatment of B16M-F10 cells with a bcl-2–AS, when combined with L-buthionine-(S,R)-sulfoximine (a specific GSH synthesis inhibitor)–induced GSH depletion, promoted massive metastatic cell death within hepatic microvessels and abrogated tissue invasion (21).

The thiol redox status (controlled by GSH) is one of the physiologic effectors regulating the mitochondrial permeability transition pore complex (35). Mitochondria are highly dependent on GSH for the prevention of oxidative damage from aerobic respiration, but are incapable of GSH synthesis and must transport cytosolic GSH across the outer membrane (36). GSH uptake into B16M-F10 mitochondria reflects function of a multicomponent transport system (25, 37). Measurement of GSH uptake at very low cytosolic GSH levels (0.1-1 mmol/L, which can only be expected in malignant cells under conditions of severe, e.g., drug-induced, intracellular GSH depletion) revealed a high-affinity component with an apparent Km of 88 ± 17 µmol/L (25). At higher external GSH levels (2-9 mmol/L), a low-affinity component transports GSH with an apparent Km of 4.5 ± 0.66 mmol/L (25). Thus, inhibition of GSH transport into mitochondria by excess cytosolic Glu levels (as those induced by a GED, see above), if complemented with G3139 + verapamil + acivicin to deplete the total cellular GSH pool, would render the cancer cells more susceptible to chemotherapy and/or radiation and to TNF-{alpha}.

Bcl-2 depletion confers multiple benefits in the treatment strategy presented here due to inherent inhibition of apoptosis via interactions with other Bcl-2 family members and indirect effects on GSH status. Our results show that G3139 administration reduces Bcl-2 levels in metastatic cells isolated from liver (Fig. 1B) and metastases volume by 72% (MV, Table 2). Combined G3139 + verapamil + acivicin treatment decreases MV by 83% (Table 2). More complex treatments were capable of even more significant reductions, but metastatic cells were detectable in all groups except when animals received the full treatment regimen (Table 2). Failure to eliminate all metastatic cells was confirmed by short survival durations (Fig. 3). These failures may reflect the natural heterogeneity of Bcl-2 and GSH levels in a cancer cell population in vivo and the rapid regrowth of surviving cancer cells during or after therapy (38). Surviving invasive cells may benefit from metastatic mechanisms already induced by oxidative stress, including activation of early growth response-1 transcription factor gene and metalloproteases and increased expression of cell-adhesion molecules, manganese-containing superoxide dismutase and catalase activities (9), and other key invasive growth-related molecules such as vascular endothelial growth factor-A, HIF-1, and protein 8.5 Only the combination treatment of G3139 + verapamil + acivicin + paclitaxel + TNF-{alpha} + IFN-{gamma} and X-rays was capable of eliminating the invasive metastatic cell population (Table 2). The absence of surviving cells was confirmed by very long-term survivors who remained free of tumors (Fig. 3).

Can clinical applications be derived from these results?

G3139 has been evaluated in multiple clinical trials and is well tolerated at doses comparable to those used in this study (e.g., ref. 14 and references therein).

In preclinical studies, administration of a GED decreased weight loss and did not accelerate tumor growth (e.g., ref. 9). In clinical trials, patients receiving Gln-supplemented parenteral nutrition during chemo- and radiotherapy had improved nitrogen balance, diminished incidence of infections, and less extracellular fluid accumulation. These clinical measures are all consistent with the role of Gln in stimulating protein synthesis in skeletal muscle, enhancing immune functions, and supporting endothelial function and integrity (39). In addition, Gln supplementation might have reduced treatment toxicities (39).

Verapamil is a calcium ion influx inhibitor frequently used for the management of hypertension and angina pectoris, which has been also used in patients receiving chemotherapy for, e.g., myeloma or acute lymphocytic leukemia. Cells isolated from these patients showed increased accumulation of daunorubicin or vincristine (40) when patient plasma concentrations of verapamil were similar to murine verapamil plasma levels in our experiments (8).

Acivicin has been evaluated for antitumor activity in phases I and II clinical trials because expression of {gamma}-GT was observed in melanoma and cancers of the liver, lung, breast, and ovary (23, 41). These trials revealed central nervous system toxicities, so a maximum dose of 50 mg acivicin/m2/day in combination with the amino acid solution aminosyn to reduce acivicin uptake in the central nervous system was proposed. However, the application of acivicin described here is not predicted to require aminosyn. The pharmacokinetic parameters of acivicin in patients (41), our efficacy results, and previous preclinical studies (8) all suggest that acivicin plasma levels sufficient to block {gamma}-GT activity in tumors would be below the levels that resulted in CNS toxicity.

High TNF-{alpha} doses can cure tumor-bearing mice, but lead to injury of normal tissues and can cause a lethal shock syndrome (42). Such toxicity limits the clinical use of high-dose TNF-{alpha}. In medical practice, high-dose TNF-{alpha} plus chemotherapy, with or without IFN-{gamma}, can be administered regionally through isolated limb perfusion. This procedure may produce, e.g., between 70% and 80% complete remission in cases of in-transit melanoma metastases and between 25% and 36% complete remission in cases of unresectable soft-tissue sarcomas (43). Thus, approaches that could potentiate the effect of TNF-{alpha} in vivo, without causing severe toxicity, would be beneficial for the treatment of systemically disseminated malignant tumors. The doses of rmTNF-{alpha} (2 x 104 units/µg/day per mouse) and rmIFN-{gamma} (4 x 103 units/day per mouse) are within a clinically acceptable range when compared with doses previously administered to melanoma-bearing patients (e.g., 0.5-4 mg of TNF-{alpha} and 1.5 x 106 units of IFN-{gamma}; ref. 44). Our experimental strategy incorporates cytokines to eliminate the final subset of invasive cells (Table 2) with acceptable toxicities (Table 3).

The X-ray dose (5 Gy) used for the experiments displayed in Table 2 is below the LD50 reported for mice (~7.5-8 Gy; e.g., ref. 19). The LD50 in humans from acute, whole-body radiation exposure is ~4 to 5 Gy (45). Nevertheless, in patients, after image diagnosis (using, e.g., axial tomography and/or positron emission techniques), radiations can be concentrated in tumor foci; moreover, the human dose can be scaled up if bone marrow transplantation follows radiation/chemotherapy-induced aplasia. Paclitaxel dose (5 mg/kg) is also within a clinically acceptable range. For instance, in patients with metastatic carcinoma of the breast, 175 mg paclitaxel/m2 (~4.2-4.5 mg/kg) administered i.v. over 3 h every 3 weeks has been shown effective.6

In conclusion, combined Bcl-2 and GSH depletion is a powerful approach to sensitize metastatic melanoma cells to biotherapy, chemotherapy, and/or radiation. All components in this experimental therapy were administered at clinically tolerated doses. Therefore, the application of this approach to humans seems feasible. This may help to improve the poor prognosis of melanoma patients and possibly of patients bearing other malignant tumors showing similar molecular characteristics.


   Acknowledgments
 
This manuscript is dedicated to the wonderful people of the Paediatric Oncology Service at La Fe Hospital (Valencia, Spain), whose work has saved many lives.


   Footnotes
 
Grant support: Ministerio de Educación y Ciencia (SAF 2003-1886, SAF2006-2049, and AGL2005-00831) and the Generalitat Valenciana (ACOMP06/247; Spain). S. Mena and M. Benlloch held fellowships from the Ministerio de Educación y Ciencia.

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.

Competing interests statement: All authors declare that they have no competing financial interests, with the exception of B.D. Brown, who is an employee of Genta Incorporated.

4 See www.fda.gov. Back

5 Estrela et al., unpublished observation. Back

6 See, e.g., www.fda.gov. Back

Received 11/ 2/06; revised 2/12/07; accepted 2/19/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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