An endothelin receptor B antagonist inhibits growth and induces cell death in human melanoma cells in vitro and in vivo

Abstract

Activation of the endothelin receptor B (ETRB) in cultured melanocyte precursors promotes cell proliferation while inhibiting differentiation, two hallmarks of malignant transformation. We therefore tested whether ETRB has a similar role in malignant transformation of melanoma. When tested in culture, we find that the selective ETRB antagonist BQ788 can inhibit the growth of seven human melanoma cell lines, but not a human kidney cell line. This inhibition often is associated with increases in pigmentation and in the dendritic shape that is characteristic of mature melanocytes. In three cell lines we also observe a major increase in cell death. In contrast, the endothelin receptor A (ETRA) antagonist BQ123 does not have these effects, although all the cell lines express both ETRA and ETRB mRNA. Extending these studies in vivo, we find that administration of BQ788 significantly slows human melanoma tumor growth in nude mice, including a complete growth arrest in half of the mice treated systemically. Histological examination of tumor sections suggests that BQ788 also enhances melanoma cell death in vivo. Thus, ETRB inhibitors may be beneficial for the treatment of melanoma.

Figures

Figure 1

The ETRB antagonist BQ788 induces morphological changes in cultured melanoma cells. Cells were cultured for 4 days with 100 μM BQ788 (A and C) or with vehicle (B and D). The cell lines were melanoma line SK-MEL 28 (A and B) and melanoma line SK-MEL 5 (C and D). Note the different shape of the drug-treated melanoma cells and their accumulation of black pigment. Pictures were taken with bright-field optics at ×40 magnification.

Figure 2

BQ788 reduces the number of viable cells in cultured melanoma but not kidney cells. Cells were cultured in the presence of the antagonist, vehicle, or with no treatment for 4 days. Three wells of each condition were subjected to the MTS assay. The mean values were calculated and plotted as the percentage of the vehicle-treated value ± SEM. The experiment was repeated three times, with similar results. BQ788 values are indicated by ∗ when significantly different from controls (P < 0.05).

Figure 3

Reverse transcription–PCR reveals the expression of ETRB and ETRA mRNA in all cell lines used. Bands corresponding to ETRB (A) (220 bp) and ETRA (B) (352 bp) mRNAs were obtained by using published primer sequences and protocols (18). For the cell line RPMI 7951, an additional five PCR cycles were needed to obtain a strong band for the ETRB shown in A. Molecular weight markers (m) are shown in the outer lanes of the gel near the negative controls, in which the cDNA was omitted.

Figure 4

The selective ETRA antagonist BQ123 does not reduce the number of viable melanoma cells in culture. Conditions and presentation are as described for the BQ788 experiment shown in Fig. 2.

Figure 5

An ETRB-selective agonist abrogates the effects of BQ788 on melanoma cells. In experiments similar to those illustrated in Fig. 2, S6c (0.1–25 μM) was tested for its effects on A375 cells cultured for 4 days alone or in combination with BQ788 (75 μM). The ETRB agonist stimulates cell growth and blocks the inhibitory effect of the ETRB antagonist. The means of absolute values (n = 3–5) were calculated ± SEM and plotted. P values were calculated by using the Student’s t test and are indicated by ∗ when significantly different (P < 0.05) from controls or in comparison with BQ788 values when the addition of S6c to BQ788 was tested.

Figure 6

Intratumor injection of BQ788 inhibits melanoma tumor growth in nude mice. Nude mice (nu/nu, BALB/c background) were implanted with grafts of A375 cells s.c. in the flank. After the tumors had reached approximately 4 mm in diameter they were injected daily with BQ788 for 9 days. Perpendicular tumor diameters were measured daily to estimate tumor volume. Controls were injected on the same schedule with vehicle. Data from three experiments using 10 BQ788-treated mice and 8 vehicle-treated mice are pooled. P values were calculated by using the Student’s t test and are indicated by ∗ when significantly different from controls (P < 0.05). Similar conclusions come from measuring tumor weights, as described in the text.

Figure 7

Systemic administration of BQ788 inhibits melanoma tumor growth in nude mice. The experiments were similar to those described in Fig. 6, except that the drug and vehicle were injected daily i.p. In A, the data for the six BQ788-treated mice were pooled. In B, separate curves are drawn for the data from the three mice in which BQ788 slowed tumor growth and for the data from the three mice in which BQ788 caused the tumors to regress. P values were calculated by using the Student’s t test and are indicated by ∗ when significantly different from controls (P < 0.05).

Figure 8

TUNEL staining of A375 cell tumors grown in nude mice reveals evidence for BQ788-stimulated apoptosis. Representative sections are illustrated from four tumors from BQ788-injected (i.p.) mice and three tumors from vehicle-injected mice. TUNEL-positive cells are in most cases much more frequent in tumors from the drug-treated animals, suggesting enhanced apoptosis. (A–D) BQ788-treated mice. (A and B) Less-affected tumors. (C and D) Strongly affected tumors. (E–G) Vehicle-treated mice. Pictures were taken at ×20 magnification.