Maraviroc/cisplatin combination inhibits gastric cancer tumoroid growth and improves mice survival

Abstract

Background: Gastric cancer (GC) is a significant cancer-related cause of death worldwide. GC's most used chemotherapeutic regimen is based on platinum drugs such as cisplatin (CDDP). However, CDDP chemoresistance reduces the survival rate of advanced GC. The immune C-C chemokine receptor type 5 (CCR5) have been proposed as a pivotal factor in cancer progression since its blockade has been linked with antineoplastic effects on tumor cell proliferation; nevertheless, its role in the chemoresistance of GC has not been elucidated. This study aimed to determine the effects induced by the CCR5 using Maraviroc (MVC), a highly selective CCR5 antagonist, on CDDP-resistant AGS cells (AGS R-CDDP), tumoroids (3D tumor spheroids), and animal models.

Results: The combined CDDP and MVC treatment reduced cell viability and inhibited tumoroid formation in AGS R-CDDP cells. The effects of the MVC/CDDP combination on apoptosis and cell cycle progression were correlated with the increase in CDDP (dose-dependent). The mRNA levels of C-C Motif Chemokine Ligand 5 (CCL5), the main ligand for CCR5, decreased significantly in cells treated with the MVC/CDDP combination. MVC in the MVC/CDDP combination improved the survival rate and biochemical parameters of CDDP-treated mice by reducing the side effects of CDDP alone.

Conclusions: This finding suggests that MVC/CDDP combination could be a potential complementary therapy for GC.

Keywords: C-C chemokine receptor type 5 (CCR5); C-C motif chemokine ligand 5 (CCL5); Chemoresistance; Cisplatin (CDDP); Gastric cancer (GC); Maraviroc (MVC).

Fig. 1

Cell Viability of AGS R-CDDP cells exposed to MVC and/or CDDP. Cells were incubated with MVC and/or CDDP for 24, 48, and 72 h, and cell viability was measured using a standard viability assay (MTT). Two-way ANOVA with Bonferroni post-hoc test was used to compare the groups. Values of P ≤ 0.05 were considered statistically significant. *P ≤ 0.05, ***P < 0.001 and ***P < 0.0001. Data were expressed as mean ± SD of three biological replicates. Maraviroc (MVC) and Cisplatin (CDDP)

Fig. 2

Percentage of cell death of AGS R-CDDP cells exposed to MVC and/or CDDP. The results include annexin V-positive/PI-negative cells at Q4 coordinate, annexin V/PI double-positive cells at Q2, and annexin V-negative/PI-positive cells at Q1. Cells were incubated for 72 h with MVC and/or CDDP, and the cell death was measured by flow cytometry. The fluorescence was read at maximum excitation/emission of 499⁄521 for Alexa Fluor 488 annexin V and 535⁄617 for PI. Kruskal-Wallis with Dunn post-hoc test was used to compare groups. Values of P ≤ 0.05 were considered statistically significant. ***P < 0.001 and ****P < 0.0001. Data were expressed as mean ± SD of three biological replicates. Maraviroc (MVC) and Cisplatin (CDDP)

Fig. 3

Cell cycle analysis of AGS R-CDDP cells exposed to MVC and/or CDDP. Cells were incubated with MVC and/or CDDP for (A) 24, (B) 48, and (C) 72 h by flow cytometry. Two-way ANOVA with Bonferroni post-hoc test was used to compare the groups. Data were expressed as mean ± SD of three biological replicates. Maraviroc (MVC) and Cisplatin (CDDP)

Fig. 4

Relative expression of CCL5 in AGS R-CDDP exposed to MVC and/or CDDP. Cells were incubated with MVC and/or CDDP for 72 h, and mRNA expression was quantified by qRT-PCR using the 2^−ΔΔCT method. ACTB was used as the reference gene. The Kruskal-Wallis test with the Dunn post-hoc test was used to compare groups. Values of P ≤ 0.05 were considered statistically significant. **P < 0.01. Data were expressed as mean ± SD of three biological replicates. Maraviroc (MVC) and Cisplatin (CDDP)

Fig. 5

Tumoroid formation. (A) Scheme of development of tumoroids from AGS-RCDDP cells. (B) Representative images of tumoroids (3D tumor spheroids) on day 24. (C) Tumoroids number after treatments with MVC and/or CDDP. (D) Tumoroids size after treatments with MVC and/or CDDP. Tumoroids with a size > 100 μm were quantified. The size bar corresponds to 100 μm. Tumoroid formation and size were analyzed using one-way ANOVA with Tukey´s multiple comparisons post-hoc test. Values of P ≤ 0.05 were considered statistically significant. *P ≤ 0.05, **P < 0.01, ****P < 0.0001. Data were expressed as mean ± SD of three biological replicates. Maraviroc (MVC) and Cisplatin (CDDP)

Fig. 6

In vivo assay. A) Schematic subcutaneous tumor formation in BalbC NOD/SCID mice from tumoroids. (B) Tumor volumes of animals treated with MVC and/or CDDP were measured on day 21. (C) Time course of tumor formation in animals treated with MVC and/or CDDP. Mixed-effect analysis followed by Tukey’s multiple comparison test was used (*P ≤ 0.05, **P < 0.01). Data were expressed as mean ± SEM of three biological replicates) D) Survival chart of animals treated with MVC and/or CDDP. The Log-rank (Mantel-Cox) test was used to assess animal survival. The overlap of the survival curves for the control group, the group treated with MVC, and the group treated with the MVC/CDDP combination indicates that there were no deaths before euthanasia, in contrast to the group treated with CDDP alone. Values of P ≤ 0.05 were considered statistically significant. Maraviroc (MVC) and Cisplatin (CDDP)