Irinotecan and its metabolite SN38 inhibits procollagen I production of dermal fibroblasts from Systemic Sclerosis patients

Abstract

Systemic sclerosis (SSc) is a rare autoimmune connective tissue disease characterized by a microangiopathy and fibrosis of the skin and internal organs. No treatment has been proved to be efficient in case of early or advanced SSc to prevent or reduce fibrosis. There are strong arguments for a key role of topo-I in the pathogenesis of diffuse SSc. Irinotecan, a semisynthetic derivative of Camptothecin, specifically target topo-I. This study was undertaken to evaluate the effects of noncytotoxic doses of irinotecan or its active metabolite SN38 on collagen production in SSc fibroblasts. Dermal fibroblasts from 4 patients with SSc and 2 healthy donors were cultured in the presence or absence of irinotecan or SN38. Procollagen I release was determined by ELISA and expression of a panel of genes involved in fibrosis was evaluated by qRT-PCR. Subcytotoxic doses of irinotecan and SN38 caused a significant and dose-dependent decrease of the procollagen I production in dermal fibroblasts from SSc patients, respectively - 48 ± 3%, p < 0.0001 and - 37 ± 6.2%, p = 0.0097. Both irinotecan and SN38 led to a global downregulation of genes involved in fibrosis such as COL1A1, COL1A2, MMP1 and ACTA2 in dermal fibroblasts from SSc patients (respectively - 27; - 20.5; - 30.2 and - 30% for irinotecan and - 61; - 55; - 50 and - 54% for SN38). SN38 increased significantly CCL2 mRNA level (+ 163%). The inhibitory effect of irinotecan and its active metabolite SN38 on collagen production by SSc fibroblasts, which occurs through regulating the levels of expression of genes mRNA, suggests that topoisomerase I inhibitors may be effective in limiting fibrosis in such patients.

Figure 1

Analysis of irinotecan effect on procollagen I release of dermal SSc fibroblasts. The level of procollagen I release was evaluated after 48 h in dermal fibroblasts from SSc patients incubated with irinotecan. A comparison of procollagen I release level was done between non-treated condition and treated conditions. (A) Data from a representative experiment showing a dose-dependent effect on the procollagen I production of SSc fibroblasts after incubation with 4 different doses of irinotecan (i.e. 0.006 µg/ml, 0.6 µg/ml, 6 µg/ml, 40 µg/ml). (B) Cumulative data from 3 independent experiments showing a decrease of procollagen I production in SSc fibroblasts incubated with irinotecan (40 µg/ml). ****p < 0.0001 using paired t-test.

Figure 2

Analysis of SN38 effect on procollagen I release of dermal SSc fibroblasts. The level of procollagen I release was evaluated after 48 h in dermal fibroblasts from SSc patients incubated with SN38. A comparison of procollagen I release level was done between non-treated condition and treated conditions. (A) Data from a representative experiment showing a dose-dependent effect on the procollagen I production of SSc fibroblasts after incubation with 4 different doses of SN38 (i.e. 0.01 µg/ml, 0.06 µg/ml, 0.1 µg/ml, 0.3 µg/ml). (B) Cumulative data from 3 independent experiments showing a decrease of procollagen I production in SSc fibroblasts incubated with SN38 (0.3 µg/ml). ** p < 0.01 using paired t-test.

Figure 3

Analysis of irinotecan effect on mRNA expression of genes involved in fibrosis in dermal SSc fibroblasts. Relative mRNA expression of a panel of genes involved in fibrosis (from A to F: COL1A1, COL1A2, MMP1, TIMP1, CCL2 and ACTA2) in dermal fibroblasts obtained from 4 systemic sclerosis patients treated with irinotecan (20 µg/mL), in comparison to a non-treated condition. The non-treated condition was arbitrary fixed at 100%. Analysis of qRT-PCR reactions normalized to stable housekeeping gene GAPDH (Glyceraldehyde-3-phosphate dehydrogenase). A level above 100% corresponds to a gene overexpression in the treated condition and a level below 100% corresponds to a gene downregulation in the treated condition. Mean ± SEM, ns: non significant, * p < 0.05 using paired t-test.

Figure 4

Analysis of SN38 effect on mRNA expression of genes involved in fibrosis in dermal SSc fibroblasts. Relative mRNA expression of a panel of genes involved in fibrosis (from A to F: COL1A1, COL1A2, MMP1, TIMP1, CCL2 and ACTA2) in dermal fibroblasts obtained from 4 systemic sclerosis patients treated with SN38 (0.4 µg/mL), in comparison to a non-treated condition. The non-treated condition was arbitrary fixed at 100%. Analysis of qRT-PCR reactions normalized to stable housekeeping gene GAPDH (Glyceraldehyde-3-phsophate dehydrogenase). A level above 100% corresponds to a gene overexpression in the treated condition and a level below 100% corresponds to a gene downregulation in the treated condition. Mean ± SEM, ns: non significant, *p < 0.05, ** p < 0.01, *** p < 0.001 using paired t-test.