The inhibitory effects of camptothecin, a topoisomerase I inhibitor, on collagen synthesis in fibroblasts from patients with systemic sclerosis

Abstract

The main manifestation of systemic sclerosis (SSc) is the overproduction of extracellular matrix, predominantly type I collagen. This study was undertaken to evaluate the effects of noncytotoxic doses of the topoisomerase I inhibitor camptothecin (CPT) on collagen production in the activated dermal fibroblasts from patients with SSc and healthy donors. The fibroblasts were cultured in the presence or absence of CPT. Production of collagenous proteins by fibroblasts was determined in cell and matrix layers by ELISA and in conditioned media by [(3)H]proline incorporation, gel electrophoresis, and autoradiography. Expression of alpha2(I) collagen (COL1A2) mRNA was measured by northern blot, and the activity of COL1A2 promoter was determined by a chloramphenicol acetyltransferase assay. CPT (10(-7) M) decreased the deposition of type I collagen by 68%, of type III by 38%, and of type VI by 21% in SSc fibroblasts and to a lesser degree in healthy controls. Similarly, CPT (10(-8) M to 10(-6) M) significantly inhibited secretion of newly synthesized collagenous proteins into conditioned media by 50%. CPT (10(-8) M to 10(-6) M) caused a significant dose-dependent inhibition of COL1A2 mRNA levels and COL1A2 promoter activity, both by as much as 60%. The inhibitory effect of CPT on collagen production by fibroblasts from patients with SSc suggests that topoisomerase I inhibitors may be effective in limiting fibrosis in such patients.

Figure 1

The effects of CPT on deposition of collagen types I (a), III (b), and VI (c) and elastin (d) in SSc and healthy control fibroblasts. Fibroblasts were grown to confluence in 96-well plates and then incubated for 24 h with CPT in DMEM/1% FBS. In control wells ('None'), the CPT was omitted. ELISA was performed on the cell and matrix layers with appropriate antibodies as described in the Patients and methods section. Bars show the relative amounts of collagen types I, III, and VI (a–c) from 11 pairs of SSc and healthy fibroblast cell lines and of elastin (d) from from four pairs of SSc and healthy fibroblast cell lines. All experiments were done in triplicate. CPT = camptothecin; FBS = fetal bovine serum; SSc = systemic sclerosis. ^*P < 0.05, ^**P < 0.005.

Figure 2

CPT inhibits secretion of collagenous proteins by both SSc and healthy control fibroblasts.Confluent fibroblasts in 12-well plates were incubated for 48 h in 0.1% BSA/DMEM containing ascorbic acid (50 μg/ml), and then treated for 24 h with various concentrations of CPT (none, or 10^-9, 10^-8, 10^-7, or 10^-6 mol/l). After CPT treatment, 20 μCi/ml of [³H]proline was added to the medium for an additional 24 h. Aliquots of conditioned media normalized for cell number were loaded on a 6% SDS–polyacrylamide gel. After electrophoresis, the gel was enhanced by fluorography and visualized by autoradiography. (a) Representative autoradiographs from SSc and healthy control fibroblasts. (b) Summary of quantitative analysis of the collagenous proteins expressed by SSc (left panel) and healthy control fibroblasts (right panel) after CPT treatment. Bars show means ± SEM from 10 (SSc) and 9 (healthy) independent experiments. The steady-state level of collagenous proteins in CPT-free controls ('None') was arbitrarily set at 1. (c) Representative autoradiogram of the total protein synthesis in the SSc fibroblasts after CPT treatment. Similar results (not shown) were obtained with the control cells. BSA= bovine sreum albumin; CPT = camptothecin; SSc = systemic sclerosis. ^*P < 0.05, ^**P < 0.005).

Figure 3

CPT inhibits expression of COL1A2 mRNA in SSc and healthy control fibroblasts. SSc and healthy control cells were incubated with various concentrations of CPT (10^-9 to 10^-6 mol/l) and analyzed by northern blotting. (a) Representative northern blot. (b, c) Summary of quantitative analysis of northern blots from healthy control and SSc cell lines, respectively. All values were corrected for loading differences as determined by intensity of GAPDH mRNA. Values from the CPT-treated cells indicate band density relative to that of untreated cells ('None'), which was set at 100. Bars show means ± SEMof the COL1A2/GAPDH ratio from 13 independent experiments using seven different pairs of SSc and healthy control fibroblasts. COL1A2 = type α2(I) collagen; CPT = camptothecin; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; SSc = systemic sclerosis. ^*P < 0.01, ^**P < 0.005.

Figure 4

CPT inhibits COL1A2/CAT promoter activity. Human dermal fibroblasts were transiently transfected with 20 μg of COL1A2/CAT promoter construct and treated with CPT (at 10^-12 to 10^-6 mol/l) for 24 h. Samples of cell extracts were normalized for protein concentration. Comparisons of the promoter transcription activity after CPT treatment were made between unstimulated and CPT-treated cells (done in duplicate). Transcriptional activity of the COL1A2/CAT gene in the CPT-free control ('None') was arbitrarily set at 100. Bars show mean ± SEM from 11 independent experiments (^*P < 0.01). COL1A2 = type α2(I) collagen; CAT = chloramphenicol acetyltransferase; CPT = camptothecin.