Neutralizing tumor-related inflammation and reprogramming of cancer-associated fibroblasts by Curcumin in breast cancer therapy

Abstract

Tumor-associated inflammation plays a vital role in cancer progression. Among the various stromal cells, cancer-associated fibroblasts are promising targets for cancer therapy. Several reports have indicated potent anti-inflammatory effects attributed to Curcumin. This study aimed to investigate whether inhibiting the inflammatory function of cancer-associated fibroblasts (CAFs) with Curcumin can restore anticancer immune responses. CAFs were isolated from breast cancer tissues, treated with Curcumin, and co-cultured with patients' PBMCs to evaluate gene expression and cytokine production alterations. Blood and breast tumor tissue samples were obtained from 12 breast cancer patients with stage II/III invasive ductal carcinoma. Fibroblast Activation Protein (FAP) + CAFs were extracted from tumor tissue, treated with 10 μM Curcumin, and co-cultured with corresponding PBMCs. The expression of smooth muscle actin-alpha (α-SMA), Cyclooxygenase-2(COX-2), production of PGE2, and immune cell cytokines were evaluated using Real-Time PCR and ELISA, respectively. Analyzes showed that treatment with Curcumin decreased the expression of genes α-SMA and COX-2 and the production of PGE2 in CAFs. In PBMCs co-cultured with Curcumin-treated CAFs, the expression of FoxP3 decreased along with the production of TGF-β, IL-10, and IL-4. An increase in IFN-γ production was observed that followed by increased T-bet expression. According to our results, Curcumin could reprogram the pro-tumor phenotype of CAFs and increase the anti-tumor phenotype in PBMCs. Thus, CAFs, as a component of the tumor microenvironment, are a suitable target for combination immunotherapies of breast cancer.

Figure 1

Isolation and characterization of CAFs. (a) The outgrowth of fibroblastic cells from tumor tissue began on day two and reached 80% confluence on day 21. These cells were passaged and maintained for Curcumin treatment and co-culture experiments. (b) Passage 2 or 3 CAFs were used in all experiments. Fibroblastic colonies were cultured in 4-well plates and immune stained with rabbit anti-human FAP, and (c) nuclei were counterstained with DAPI. Cells showed expression of FAP.

Figure 2

IC₅₀ value of Curcumin in 3 isolated CAFs. CAFs were cultured in standard conditions and treated with different concentrations of Curcumin. Log(inhibition)-curve fitting model was used to calculate the IC₅₀ value. Data represents the mean of triplicates of 3 isolated CAFs. Results showed that the IC₅₀ = 15.15 μM ± 1.15 with an R² of 0.8591.

Figure 3

Effect of Curcumin on CAF phenotype. (a) The effect of Curcumin treatment on the mRNA expression of COX-2 and αSMA was analyzed using Real-Time PCR. Results showed that CUR could decrease the functional markers associated with CAF phenotype, COX-2 and αSMA. (b) Each sample's gene expression fold changes were analyzed using the paired T-test in three replicates. Individual comparisons also showed a significant reduction in the expression of COX-2 and α-SMA. Data represents mean ± SD. Significant changes are indicated with asterisk *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

Figure 4

Effect of Curcumin on CAFs' cytokine production. Production of cytokines IL-10, TGF-β, and PGE2 was evaluated using ELISA. Results indicated that after treatment with Curcumin, the production of cytokines associated with CAF phenotype and function, including TGF- β, was significantly reduced. The production of PGE2 from Curcumin-treated CAFs also showed a significant decrease. However, the level of IL-10 was unaffected. Data represents mean ± SD. Significant changes are indicated with asterisk *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

Figure 5

Increased Stimulation Index (SI) of PBMCs co-cultured with CAFs treated with Curcumin. The stimulation index of PBMCs co-cultured with corresponding CAFs and their respective control groups indicated that treatment with Curcumin restored the proliferation of PBMCs. However, this increase was not statistically significant. The highest SI was observed in the 1:5 ratio of CAF: PBMC co-cultures. As shown, in the no-treatment group, the highest SI corresponds to the lowest ratio, indicating the suppressive effects of CAFs. Data represents mean ± SD. Significant changes are indicated with asterisk *P-value < 0.05,

Figure 6

PBMCs gene expression analysis. The expression of transcription factors associated with Th differentiation, including FOXP3, T-BET, and GATA-3, was analyzed using Real-Time PCR. (a) Results indicated that treatment with Curcumin increased the expression of markers associated with Th1, including T-BET, and decreased the expression of markers associated with Tregs (FoxP3). (b) Individual changes in gene expression were analyzed with the paired-T test of 3 replicates. Results showed that treatment with Curcumin increased the expression of Tbet in all treated co-cultures efficiently. Data represents mean ± SD. Significant changes are indicated with asterisk *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

Figure 7

Cytokine production in co-cultures of PBMCs and Curcumin-treated CAFs. CAFs were isolated and treated with Curcumin. PBMCs were added, and the levels of cytokines were assessed in the supernatant after 48 h (a). The production of cytokines associated with Th differentiation, including IL-4, PGE2, TGF-β, IL-10, and IFN-γ (b–f), was evaluated in culture supernatants using ELISA. Results indicated that treatment with Curcumin increased the production of IFN-γ and reduced the production of TGF-βand IL-10, indicative of increased Th1 and inhibited Treg phenotype. Additionally, no significant change in PGE2 was observed. Data represent mean ± SD. Significant changes are indicated with asterisk *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.