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IMMUNOBIOLOGY: ARTICLES

Tacrolimus enhances transforming growth factor-β1 expression and promotes tumor progression

Maluccio, Mary1; Sharma, Vijay2; Lagman, Mila2; Vyas, Shefali2; Yang, Hua2; Li, Baogui2; Suthanthiran, Manikkam2 3

Author Information
doi: 10.1097/01.TP.0000081399.75231.3B
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Abstract

Transforming growth factor (TGF)-β1, a pleiotropic cytokine and secretory product of several cell types, has been implicated in many aspects of cancer biology including heightened invasiveness and metastatic progression (1,2). The incidence and aggressiveness of certain malignancies are increased in organ graft recipients. Immunosuppressive drugs, by virtue of their ability to impair host immunosurveillance mechanisms, are considered to be major risk factors for the heightened incidence and invasiveness of these malignancies (3–6).

In an earlier study, we identified a novel and host immunity-independent mechanism for the immunosuppressive drug cyclosporine-associated tumor progression (7). We found that cyclosporine conditioning of tumor cells conferred an invasive phenotype by a cell-autonomous mechanism and promoted neoplastic progression in T-cell, B-cell, and natural killer (NK)-cell–deficient severe combined immunodeficient (SCID)-beige mice. We also found that TGF-β1 blockade prevented cyclosporine-associated increase in the number of pulmonary metastases in the SCID-beige mice.

Cyclosporine and tacrolimus are believed to exert their immunosuppressive effects through targeted binding and inactivation of calcineurin, a calcium- and calmodulin-dependent serine and threonine phosphatase (8,9). Cyclosporine and tacrolimus have been shown to share not only immunosuppressive mechanisms but also toxic side effects. Indeed, preclinical and clinical data exist indicating that tacrolimus, in a similar fashion to cyclosporine, is associated with renal fibrosis (10,11).

We reported that cyclosporine induces TGF-β1 overexpression in multiple cell types (12,13). We also observed that cyclosporine enhances in vivo expression of TGF-β1 in mice and humans (14,15). Khanna et al. reported that in vitro conditioning of human T cells or human A-549 adenocarcinoma cells with tacrolimus results in the overexpression of TGF-β1 (16). Because TGF-β1 can enhance tumor invasiveness and metastasis (1,2), and because tacrolimus may share with cyclosporine the property of inducing TGF-β1 in vivo, we explored the in vivo effect of tacrolimus on tumor progression.

In the current investigation, we used a well-established mouse renal cancer cell (RCC) pulmonary metastasis model (17) that shares several similarities with the clinical presentation of renal cancer to explore the effect of tacrolimus. Tacrolimus is a potent immunosuppressant and can impair host immunosurveillance barriers to tumor progression. To investigate the effect of tacrolimus on tumor progression independent of its effects on adaptive immunity, we also examined the effect of tacrolimus on tumor progression in T-, B-, and NK-cell–deficient SCID-beige mice (18). In view of the potential contribution of TGF-β1 overexpression to tumor progression, we investigated whether tacrolimus induces TGF-β1 overexpression in mice.

MATERIALS AND METHODS

Mice and Reagents

Inbred male BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA) and were used at 6 to 8 weeks of age. Male SCID-beige mice were purchased from Taconic Laboratories (Germantown, NY) and were used at 6 to 8 weeks of age. Tacrolimus, 2 mg/kg or 4 mg/kg, was administered by subcutaneous route every other day from 1 day before tumor inoculation to the day the mice were killed.

Tumor Cells

Murine RCCs of spontaneous origin in BALB/c mice were provided by R.H. Wiltrout, National Cancer Institute, Bethesda Maryland. RCCs were maintained by in vivo serial passages in syngeneic BALB/c mice, as described (17). The in vitro effect of tacrolimus on TGF-β1 protein secretion by RCCs was examined by plating 200,000 RCCs per well in a six-well plate for 48 hr, replacing the culture medium with protein-free medium supplemented with or without tacrolimus, collecting the cell-free supernatants, and assaying for TGF-β1 protein with the use of an isoform-specific sandwich enzyme-linked immunosorbent assay (ELISA) (19).

Pulmonary Metastasis Model

Intrarenal RCC tumors were excised under sterile conditions, and single-cell suspensions were prepared by mechanical dissociation and filtration of the resulting tumor cell suspension through a 100-μm mesh filter. Tumor cells were analyzed for number and viability using a hemocytometer. RCCs (50,000–100,000 viable tumor cells in 0.5 mL of phosphate-buffered saline) were injected in the tail vein of 6- to 8-week-old BALB/c mice or SCID-beige mice to produce pulmonary metastases and randomly assigned to three experimental groups: (1) control untreated, (2) treatment with 2 mg/kg of tacrolimus, and (3) treatment with 4 mg/kg of tacrolimus. On days 17 to 19 after tumor inoculation, the mice were killed and the number of pulmonary metastases was determined after endotracheal insufflation of lungs with 15% India ink solution and bleaching the collected lungs in Fekete’s solution (7).

Competitive Quantitative Polymerase Chain Reaction Assay

Total RNA was isolated from RCC or spleens with the use of a commercial kit (RNeasy minikit; Qiagen, Chatsworth, CA). For each sample, 1 μg of RNA was reverse-transcribed to complementary DNA, as described (12). We designed and constructed TGF-β1 specific DNA competitor with the use of the primer 3 method (Fig. 1). The 311-base pair (bp) DNA competitor of TGF-β1 was amplified with the use of a modified antisense primer that contains the external antisense primer at its 5′ end and an 18-bp subfragment internal antisense primer at its 3′ end corresponding to sequences 958 to 975 within the naturally occurring product of polymerase chain reaction (PCR). Competitive quantitative reverse transcribed-PCR assay for TGF-B1 was performed according to our previously described methodology (12). Amplification by PCR was performed in a thermocycler (GeneAmp PCR system 9600, Perkin Elmer, CA). The PCR products were resolved by electrophoresis, stained with ethidium bromide, and scanned by laser densitometry. We quantified the concentration of naturally occurring gene transcripts by measuring the ratio of the complementary DNA band to the band of the competitor. TGF-β1 mRNA levels were expressed in attograms or femtograms of mRNA per 1 μg of total RNA.

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Figure 1:
Design and construction of transforming growth factor (TGF)-β1 DNA competitor. The 311-base pair (bp) DNA competitor template of TGF-β1 was amplified with the use of a modified antisense primer that contains the external antisense primer at its 5′ end and an 18-bp subfragment internal antisense primer at its 3′ end corresponding to sequences 958 to 975 within the naturally occurring product of polymerase chain reaction (PCR) (GenBank accession no. M13177).

Enzyme-Linked Immunosorbent Assay

ELISA kits were purchased from Endogen (Woburn, MA) and used to quantify TGF-β1 protein concentration, as described (19). Sera were diluted 10:1, and the sandwich ELISA procedure was performed using an immobilized capture antibody and a biotinylated antibody in solution phase. A standard curve for TGF-β1 was constructed, and the curve-fitting software program with the four-parameter algorithm was used to quantify TGF-β1 concentration.

Statistical Analysis

We used GraphPad Prism 3.02 statistical software for Windows (GraphPad Software, San Diego, CA). The parameters (number of pulmonary metastases, TGF-β1 mRNA levels, and TGF-β1 protein levels) used to measure the effect of tacrolimus were each used as the dependent variable in a one-way analysis of variance (ANOVA) to test for differences among the untreated mice, mice treated with 2 mg/kg tacrolimus, and mice treated with 4 mg/kg tacrolimus. The Bonferroni multiple comparison test was then used to control the risk of a type I error while comparing the parameter in three experimental groups.

RESULTS

Effect of Tacrolimus on the Level of Expression of Transforming Growth Factor-β1

We demonstrated that cyclosporine induces TGF-β1 overexpression in normal human T cells and other cells such as mink lung epithelial cells and human A549-adenocarcinoma cells (12,13). We also showed that that cyclosporine stimulates TGF-β1 expression in vivo (14,15). Khanna et al. reported that in vitro conditioning of mammalian cells with tacrolimus results in overexpression of TGF-β1 (16). It is not known whether tacrolimus enhances TGF-β1 expression in vivo.

We examined the in vitro and in vivo effects of tacrolimus on the expression of TGF-β1. The in vitro effect of tacrolimus on TGF-β1 expression by RCCs and the in vivo effect of tacrolimus on TGF-β1 expression in mice was evaluated at the mRNA level and at the protein level.

Tacrolimus-enhanced TGF-β1 expression and the magnitude of its in vitro stimulatory activity was dependent on the concentration of the drug used to treat the RCCs. Results demonstrating that treatment of RCCs increases the level of expression of TGF-β1 mRNA and protein are illustrated in Figure 2. In a representative experiment, the measurement of the expression level of TGF-β1 mRNA with the use of TGF-β1–specific DNA competitor in the competitive quantitative PCR assay showed that the level was 4,900 ag/μg of total RNA with untreated RCCs, 4,250 ag/μg after treatment of RCCs with 10 ng/mL of tacrolimus, and 8,650 ag/μg after treatment with 100 ng/mL of tacrolimus (Fig. 2A).

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Figure 2:
In vitro stimulatory effect of tacrolimus on TGF-β1 expression. (A) Murine renal cancer cells (RCCs) of spontaneous origin in BALB/c mice were incubated with or without 10 ng/mL or 100 ng/mL of tacrolimus, and total RNA were isolated from treated or untreated cells. TGF-β1 mRNA levels were measured with the use of TGF-β1–specific DNA competitor in the competitive quantitative PCR assay. TGF-β1 mRNA level was 4,900 ag/μg of total RNA with untreated RCCs, 4,250 ag/μg after treatment of RCCs with 10 ng/mL of tacrolimus, and 8,650 ag/μg after treatment of RCCs with 100 ng/mL of tacrolimus. (B) RCCs were plated at 200,000 cells per well in a six-well plate for 48 hr. The culture medium was then replaced with protein-free medium supplemented with or without the indicated concentration of tacrolimus. The cell-free supernatants were collected after incubation of RCCs with tacrolimus for 24 hr, and TGF-β1 protein concentration was measured with the use of isoform-specific sandwich enzyme-linked immunosorbent assay (ELISA). Results are from duplicate wells (mean±standard error [SE]). A standard curve for TGF-β1 was constructed, and a curve-fitting software program with the four-parameter algorithm was used to calculate TGF-β1 protein concentration.

A concentration-dependent stimulatory effect of tacrolimus on TGF-β1 protein levels was observed when cell-free supernatants, collected after incubation of RCCs with tacrolimus for 24 hr, were assayed with a TGF-β1 isoform-specific sandwich ELISA (Fig. 2B). The concentration-dependent stimulatory effect of tacrolimus was also evident with the supernatants collected after incubation of RCCs with tacrolimus for 48 hr (data not shown).

The in vivo stimulatory effect of tacrolimus on TGF-β1 expression was also dependent on the dose of tacrolimus used to treat the mice. We measured the levels of expression of TGF-β1 mRNA in the spleens from the untreated mice or the mice treated with 2 mg/kg or 4 mg/kg of tacrolimus, and the results are illustrated in Figure 3A. The mean (±standard error [SE]) level of TGF-β1 mRNA was 254±15 fg/μg in the untreated mice (n=4 mice), 283±21 fg/μg in mice treated with 2 mg/kg of tacrolimus (n=4 mice), and 412±46 fg/μg in mice treated with 4 mg/kg of tacrolimus (n=4 mice). Comparison of treatment effects in the three experimental groups showed that the null hypothesis of equal group means should be rejected (P =0.01, ANOVA). Multiple comparisons of all possible pairs of experimental groups showed that the level of TGF-β1 mRNA in mice treated with 4 mg/kg of tacrolimus is significantly higher compared with the level of TGF-β1 mRNA in the control mice (P <0.05) and in mice treated with 2 mg/kg of tacrolimus (P <0.05). The difference in the level of TGF-β1 mRNA between untreated mice and mice treated with 2 mg/kg of tacrolimus was not significant (P >0.05).

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Figure 3:
In vivo stimulatory effect of tacrolimus on TGF-β1 expression. (A) BALB/c mice were untreated or treated with 2 mg/kg or 4 mg/kg of tacrolimus, and total RNA was isolated from the spleens of untreated or treated mice. Levels of TGF-β1 mRNA (fg/μg) were measured with the use of gene-specific DNA competitor in the competitive quantitative PCR assay. The mean (±SE) level of TGF-β1 mRNA in untreated mice (n=4 mice), mice treated with 2 mg/kg of tacrolimus (n=4 mice), and mice treated with 4 mg/kg of tacrolimus (n=4 mice). P value was calculated with the use of TGF-β1 mRNA levels as the dependent variable in one-way analysis of variance (ANOVA). Bonferroni multiple comparison test showed that that the level of TGF-β1 mRNA in mice treated with 4 mg/kg of tacrolimus was significantly higher compared with the level of TGF-β1 mRNA in control mice (P <0.05) and mice treated with 2 mg/kg of tacrolimus (P <0.05). The other pairwise comparison was not significant (P >0.05). (B) BALB/c mice were untreated or treated with 2 mg/kg or 4 mg/kg of tacrolimus, and sera were collected from untreated or treated mice. Levels of TGF-β1 protein were measured with the use of isotype-specific ELISA. A standard curve for TGF-β1 was constructed, and a curve-fitting software program with the four-parameter algorithm was used to quantify TGF-β1 protein concentration. The mean (±SE) circulating level of mouse TGF-β1 in the untreated mice (n=8 mice), mice treated with 2 mg/kg of tacrolimus (n=4 mice), and mice treated with 4 mg/kg of tacrolimus (n=4 mice). P value was calculated with the use of TGF-β1 protein levels as the dependent variable in one-way ANOVA. Bonferroni multiple comparison test showed that the level of TGF-β1 protein was significantly higher in the sera of mice treated with 4 mg/kg of tacrolimus compared with the level of TGF-β1 protein in the untreated mice (P <0.01). None of the other pairwise comparisons were significant (P >0.05).

In a fashion similar to its effect on TGF-β1 mRNA expression, the extent of stimulatory effect of tacrolimus on TGF-β1 protein expression was dependent on whether the mice were treated with 2 mg/kg or 4 mg/kg body weight (Fig. 3B). The mean (±SE) circulating level of mouse TGF-β1 was 79±6 ng/mL (n=8 mice) in untreated mice, 97±10 ng/mL in mice treated with 2 mg/kg of tacrolimus (n=4 mice), and 127±7 ng/mL in mice treated with 4 mg/kg of tacrolimus (n=4 mice) (P =0.003, ANOVA). Multiple comparisons of all possible pairs of experimental groups showed that the level of TGF-β1 protein was significantly higher in the sera of mice treated with 4 mg/kg of tacrolimus compared with the level of TGF-β1 protein in the untreated mice (Bonferroni’s P <0.01). None of the other pairwise comparisons of TGF-β1 protein levels were significant (P >0.05).

Renal Cancer Cell Pulmonary Metastasis in Immunocompetent Mice Is Increased by Tacrolimus

Immunosuppressive drugs can have diametrically opposite effects on tumor progression. We reported that the calcineurin inhibitor cyclosporine promotes the progression of mouse RCC in the immunocompetent BALB/c mice (7), whereas rapamycin prevents the growth of murine RCC (20). We investigated the effect of tacrolimus, an agent that shares with cyclosporine the property of being an inhibitor of calcineurin (8,9) and shares with rapamycin the property of binding FKBP12 (21).

RCCs were injected in the tail vein of BALB/c mice and randomly assigned to the following experimental groups: (a) untreated control, (b) treatment with 2 mg/kg of tacrolimus, and (c) treatment with 4 mg/kg of tacrolimus. Tacrolimus treatment increased the number of RCC pulmonary metastases in BALB/c mice, and the results from three separate experiments are summarized in Figure 4. The mean (±SE) number of pulmonary metastases was 197±16 in the untreated mice (n=18 mice), 281±26 in the mice treated with 2 mg/kg of tacrolimus (n=17 mice), and 339±25 in the mice treated with 4 mg/kg of tacrolimus (n=21 mice). Comparison of treatment effects in the three experimental groups showed that the null hypothesis of equal group means should be rejected (P =0.0004, ANOVA). Multiple comparisons of all possible pairs of experimental groups showed that the number of pulmonary metastases in mice treated with 4 mg/kg of tacrolimus was significantly higher compared with that of control mice (Bonferroni’s P <0.001). None of the other pairwise comparisons were significant (P >0.05).

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Figure 4:
RCC pulmonary metastasis in immunocompetent mice is increased by tacrolimus. RCCs (50,000 cells in 0.5 mL of phosphate-buffered saline [PBS]) were injected in the tail vein of BALB/c mice and randomly assigned to the following treatment groups: (a) untreated control, (b) treatment with 2 mg/kg of tacrolimus, and (c) treatment with 4 mg/kg of tacrolimus. Tacrolimus was administered by the subcutaneous route and on every other day starting 1 day before tumor inoculation. The mice were killed on days 17 to 19 after tumor inoculation, and the pulmonary metastases were counted. The mean (±SE) number of pulmonary metastases in the untreated mice (n=18 mice), mice treated with 2 mg/kg of tacrolimus (n=17 mice), and mice treated with 4 mg/kg of tacrolimus (n=21 mice). Results are from three separate experiments. P value was calculated with the use of number of metastases as the dependent variable in one-way ANOVA. Bonferroni multiple comparison test showed that that the number of pulmonary metastases in mice treated with 4 mg/kg of tacrolimus was significantly higher compared with that of control mice (P <0.001). None of the other pairwise comparisons were significant (P >0.05).

Tacrolimus Increases Renal Cancer Cell Pulmonary Metastases in Severe Combined Immunodeficient-Beige Mice

Tacrolimus is a potent immunosuppressive drug, and it was not surprising that the drug increased the number of pulmonary metastases in the BALB/c mice. To test whether tacrolimus promotes tumor progression in the absence of an intact immune system, we determined the effect of tacrolimus in T-cell, B-cell and NK-cell–deficient SCID-beige mice.

RCCs were injected in the tail vein of SCID-beige mice and randomly assigned to the following experimental groups: (a) untreated control, (b) treatment with 2 mg/kg of tacrolimus, and (c) treatment with 4 mg/kg of tacrolimus. A representative experiment to illustrate the consistent increase in the number of pulmonary metastases after treatment of tumor-inoculated mice with 4 mg/kg of tacrolimus is shown in Figure 5. It is evident that treatment with 4 mg/kg of tacrolimus not only increased the number of metastases but also increased the size of tumor nodules.

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Figure 5:
Tacrolimus increases RCC pulmonary metastasis in severe combined immunodeficient (SCID)-beige mice. RCCs (100,000 cells in 0.5 mL of PBS) were injected in the tail vein of SCID-beige mice and randomly assigned to the following treatment groups: (a) untreated control, (b) treatment with 2 mg/kg of tacrolimus, and (c) treatment with 4 mg/kg of tacrolimus. Tacrolimus was administered by the subcutaneous route and on every other day starting 1 day before tumor inoculation. The mice were killed on day 17 after tumor inoculation, and the number of pulmonary metastases was counted. Treatment with 4 mg/kg of tacrolimus increased not only the number of metastases but also the size of tumor nodules.

The results from three separate experiments are summarized in Figure 6. The mean (±SE) number of pulmonary metastases was 117±18 in the untreated SCID-beige mice (n=12 mice), 137±19 in the mice treated with 2 mg/kg of tacrolimus (n=9 mice), and 216±29 in the mice treated with 4 mg/kg of tacrolimus (n=7 mice) (P <0.01, ANOVA). Multiple comparisons of all possible pairs of experimental groups showed that the number of pulmonary metastases in mice treated with 4 mg/kg of tacrolimus is significantly higher compared with the number of metastases observed in the control mice (Bonferroni’s P <0.05). None of the other pairwise comparisons of number of metastases were significant (P >0.05).

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Figure 6:
Tacrolimus increases RCC pulmonary metastasis in SCID-beige mice. RCCs (100,000 cells in 0.5 mL of PBS) were injected in the tail vein of SCID-beige mice and treated as described in the legend to Figure 5. The mean (±SE) number of pulmonary metastases in the untreated mice (n=12 mice), mice treated with 2 mg/kg of tacrolimus (n=9 mice), and mice treated with 4 mg/kg of tacrolimus (n=7 mice). Results are from three separate experiments. P value was calculated with the use of number of metastases as the dependent variable in one-way ANOVA. Bonferroni multiple comparison test showed that that the number of pulmonary metastases in the SCID-beige mice treated with 4 mg/kg of tacrolimus was significantly higher compared with that of control SCID-beige mice (P <0.05). None of the other pairwise comparisons were significant (P >0.05).

DISCUSSION

The new observation from our investigation is that tacrolimus promotes tumor progression in immunocompetent and immunodeficient mice. The tacrolimus-associated increase in the number of RCC pulmonary metastases seems to be dependent on the amount of drug administered. Tacrolimus-induced impairments in the functioning of host T cells, B cells, or NK cells does not seem to be an absolute requirement for tumor progression because the drug increased the number of pulmonary metastases in the SCID-beige mice. Our current investigation also showed that tacrolimus-associated induction of TGF-β1, a multifunctional cytokine implicated in tumor invasiveness and metastatic progression, is also dose-dependent.

Quantitative assessments of hepatic metastases and investigation of regional spread of colon tumor in experimental models found that cyclosporine enhances tumor metastasis (22,23). To the best of our knowledge, our study is the first to demonstrate that tacrolimus promotes tumor metastasis. A mechanistically interesting aspect of our observation is that tumor progression caused by tacrolimus is observed not only in the immunocompetent BALB/c mice but also in the T-cell, B-cell, and NK-cell–deficient SCID-beige mice. Our demonstration of tumor enhancement by tacrolimus in a genetically immunodeficient host challenges the idea that tumor progression caused by tacrolimus is exclusively because of the drug’s ability to impair adaptive immune effector mechanisms of the tumor-bearing host. In this regard, tacrolimus mimics cyclosporine in promoting RCC metastasis (7) and differs from rapamycin in not preventing RCC growth and spread (20)

Cell growth and polarity and cell-to-cell to physical contacts are governed by robust cellular machineries, and tumor growth, invasion, and metastatic spread are often associated with the dysregulation of the regulatory pathways. TGF-β1 has been implicated in the acquisition of tumor invasiveness and metastatic spread (1,2). High-grade and biologically aggressive tumors have been shown to contain significantly higher levels of TGF-β1 than more well-differentiated tumors, and the dynamic relationship between levels of TGF-β1 protein and TGF-β receptors is considered significant in tumor progression (1,2,24–26).

We reported that cyclosporine induces TGF-B1 expression (12–15), and that cyclosporine-associated TGF-β1 hyperexpression contributes to RCC metastasis in the SCID-beige mice (7). Cyclosporine and tacrolimus share the property of being inhibitors of calcineurin and blockers of transcriptional activation of interleukin-2 gene. It was reported that tacrolimus, in a similar fashion to cyclosporine, induces TGF-β1 expression in vitro (16). It was also reported that tacrolimus induces renal fibrosis in salt-depleted rats, and that the renal tissues display an overabundance of TGF-β1 and other proteins involved in extracellular matrix accumulation (10). It is noteworthy that in our investigation the tacrolimus-associated in vivo induction of TGF-β1 was demonstrated at the mRNA level and at the protein level. It is also noteworthy that the induction by tacrolimus occurred at the higher of the two dosages examined, and that the 4 mg/kg of tacrolimus required to demonstrate TGF-β1 induction by tacrolimus is higher than the drug dosage required to promote allograft acceptance in experimental models of transplantation (27,28).

A potential mechanism for the tacrolimus-associated tumor progression observed in our investigation is indicated by the similar dosage of tacrolimus required to induce TGF-β1 expression in vivo and to enhance RCC pulmonary metastasis. Support for the hypothesis that TGF-β1 hyperexpression contributed to metastatic spread is provided by our earlier demonstration that monoclonal antibodies directed at TGF-β prevented the cyclosporine-associated increase in the number of pulmonary metastases in SCID-beige mice (7). That blockade of TGF-β1 signaling with the use of a dominant negative TGF-β receptor prevents breast cancer metastasis to the bone in a mouse model (29) lends additional credence to the idea that an excess of TGF-β1 may be involved in tacrolimus-associated promotion of RCC metastasis.

CONCLUSION

We explored whether tacrolimus promotes RCC pulmonary metastases in the wild-type immunocompetent mouse and in the genetically immunodeficient mouse and whether tacrolimus enhances TGF-B1 expression. We also investigated whether the effects of tacrolimus on tumor progression and TGF-β1 expression are dose-dependent. Our study demonstrates that tacrolimus enhances TGF-B1 expression in vitro and in vivo, and that its stimulatory effect is dose-dependent. Tacrolimus increased the number of pulmonary metastases in a dose-dependent fashion not only in the immunocompetent wild-type mice but also in T-cell, B-cell and NK-cell–deficient SCID-beige mice. Host adaptive immunity-independent mechanisms seem to contribute to tacrolimus-associated tumor progression.

Acknowledgment.

The authors thank Dr. Thangamani Muthukumar for expert statistical assistance and Ms. Linda Stackhouse for meticulous preparation of the article.

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