Identified anti-proliferative natural compounds have no effect on

increased levels of apoptosis. To exclude the latter, HCFs treated with different anti-proliferative natural compounds were subjected to Annexin-V/7-AAD-staining and analysed by FACS. Treatment with any anti-proliferative natural compounds did not induce apoptosis in HCFs (Figure 4.13).

Figure 4.13: Investigated natural compounds do not induce apoptosis. Annexin V/7AAD staining indicating cell death induction. Neither (A) Anisomycin, (B) Geldanamycin, (C) Bufalin, (D) Gitoxigenin, (E) Piplartine nor (F) 10-Hydroycamptothecin significantly induces early or late apoptosis in HCFs.

n = 2-4 experiments. Unpaired t-test.

4.2.2 10-Hydroxycamptothecin and Piplartine treatment leads to pro-fibrotic pheno-type whereas Geldanamycin, Anisomycin, Bufalin and Gitoxigenin treatment decreases expression of fibrotic markers

Fibrosis in heart failure is characterized by various phenotypic-changes of fibroblasts. (18) Besides proliferation, expression of fibrotic markers, e.g. Collagen 1, CTGF, α-SMA and TGF-β, on mRNA or protein level plays an important role within development of cardiac fibro-sis. (16)

Results 46

Thus, anti-proliferative natural compounds optimally address repression of fibrotic markers to comprise anti-fibrotic character. Relative gene expression level was detected by rt-PCR whereas regulation on protein level was identified by western blot.

Interestingly, 24 hour treatment with anti-proliferative compound 10-Hydroxycamptothecin had no effect on collagen 1 altogether with CTGF protein level (Figure 4.14 A). In contrast, gene expression level of α-SMA, collagen 1 and collagen 3 showed promising tendencies (Figure 4.14 B).

Figure 4.14: 10-Hydroxycamptothecin presents no distinct anti-fibrotic character. Characteriza-tion of fibrotic phenotype of HCFs treated with 10-Hydroxycamptothecin. (A) Protein level analysis of collagen 1 and CTGF by western blot. 10-Hydroxycamptothecin treatment has no significant influence.

Protein expression was normalized to GAPDH. n = 3 experiments. Unpaired t-test. (B) mRNA gene expression level analysis of α-SMA, collagen 1 and 3 by real-time PCR. HCFs treated with 10-Hydroxycamptothecin exhibit significantly reduced α-SMA protein and collagen 1 protein level, as well as clear tendency of downregulated collagen 3 protein expression. Gene expression was normalized to 18S rRNA. n = 3 experiments. Unpaired t-test. * = p < 0.05.

Piplartine treatment increased protein levels of collagen 1 (Figure 4.15 A), which was paral-leled with no explicit mRNA expression regulation of fibrotic markers a-SMA, collagen 1 and 3 and CTGF (Figure 4.15 B).

Figure 4.15: Piplartine presents no distinct anti-fibrotic character. Characterization of fibrotic phe-notype of HCFs treated with Piplartine. (A) Protein level analysis of collagen 1 by western blot exhibit increased collagen 1 protein levels. Protein expression was normalized to GAPDH. n = 3 experiments.

Unpaired t-test. (B) mRNA gene expression level analysis of α-SMA, collagen 1 and 3 and CTGF by real-time PCR. HCFs treated with Piplartine exhibit only tendencies of reduced fibrotic markers. Gene expression was normalized to 18S rRNA. n = 3 experiments. Unpaired t-test.

Showing only anti-proliferative but no distinct additional anti-fibrotic effects in vitro, those two natural compounds were excluded from further analysis.

In contrast, decrease of collagen 1 protein level detected by western blot was observed in HCFs incubated with Anisomycin (Figure 4.16 A), Geldanamycin (Figure 4.16 B), Bufalin (Figure 4.16 C) and Gitoxigenin (Figure 4.16 D).

ctrl. 2,5 µM Piplartine

Results 48

Figure 4.16: Anisomycin, Geldanamycin, Bufalin and Gitoxigenin significantly decrease colla-gen 1 protein level. Influence of natural compounds on collacolla-gen 1 protein expression level detected by western blot. (A) Anisomycin, (B) Geldanamycin, (C) Bufalin and (D) Gitoxigenin treatment signifi-cantly lowers collagen 1 protein expression in HCFs. Protein expression was normalized to GAPDH. n

= 3 experiments. Unpaired t-test. * = p < 0.05.

Additionally, treatment with Anisomycin lead to decreased mRNA levels of α-SMA, collagen 1 and significant downregulation of collagen 3 mRNA level (Figure 4.17).

ctrl. 1 µM Anisomycin

Figure 4.17: Anisomycin reduces fibrotic gene expression on mRNA level. Characterization of fibrotic phenotype of HCFs treated with Anisomycin by real-time PCR. α-SMA and collagen 1 protein expression shows tendencies of repression. Collagen 3 protein level is significantly reduced. Gene expression was normalized to 18S rRNA. n = 3 experiments. Unpaired t-test. * = p < 0.05.

In contrast to protein detection by western blot, collagen 1 and 3 levels, as well as α-SMA, were significantly upregulated on mRNA gene expression level of HCFs treated with Gel-danamycin (Figure 4.18 A) suggesting impact on post-transcriptional mechanisms. However, TGF-β and CTGF gene expression levels were significantly downregulated (Figure 4.18 A), whereas detection of CTGF protein level showed no obvious effect compared to control

Results 50

Figure 4.18: Geldanamycin presents opposing fibrotic effects on mRNA and protein level. Char-acterization of fibrotic phenotype of HCFs treated with Geldanamycin. (A) mRNA gene expression analysis of fibrotic markers exhibit significantly increased α-SMA and collagen 1 and 3 protein expres-sion. TGFβ as well as CTGF gene expression is significantly reduced. Gene expression was normal-ized to 18S rRNA. n = 3 experiments. Unpaired t-test. * = p < 0.05. ** = p < 0.01. (B) CTGF protein level detected by western blot displays no difference compared to DMSO control. Protein expression was normalized to GAPDH. n = 3 experiments. Unpaired t-test.

24 hours of Bufalin treatment likewise resulted in significant reduction of TGF-β mRNA level as well as decrease of collagen 3 gene expression (Figure 4.19).

Figure 4.19: Bufalin potently represses TGFβ and collagen 3 mRNA expression level. Characteri-zation of fibrotic phenotype of HCFs treated with Bufalin by real-time PCR. α-SMA and TGFβ mRNA expression is significantly reduced. Gene expression was normalized to 18S rRNA. n = 3 experiments.

Unpaired t-test. * = p < 0.05.

ctrl. 1 µM Geldanamycin

GAPDH CTGF

B C T G F

CTGF/GAPDH (relative to control)

D M S O c t r l. 1 µ M G e ld a n a m y c in

0 .0 0 .5 1 .0 1 .5 2 .0

T G F1

TGF1/18S (relative to control)

D M S O c tr l. 1 µ M B u fa lin

0 .0 0 .5 1 .0 1 .5

*

C O L 3

COL3A1/18S (relative to control)

D M S O c tr l. 1 µ M B u fa lin

0 .0 0 .5 1 .0 1 .5

*

Treatment of HCFs with Gitoxigenin was associated with reduced α-SMA mRNA expression (Figure 4.20 A) without having an impact on CTGF protein expression (Figure 4.20 B).

Figure 4.20: Gitoxigenin significantly reduces α-SMA gene expression but has no influence on CTGF protein expression. Characterization of fibrotic phenotype of HCFs treated with Gitoxigenin.

(A) mRNA gene expression analysis of fibrotic marker α-SMA exhibit significant reduction of mRNA level. Gene expression was normalized to 18S rRNA. n = 3 experiments. Unpaired t-test. * = p < 0.05.

** = p < 0.01. (B) CTGF protein level detected by western blot exhibit no difference compared to DMSO control. Protein expression was normalized to GAPDH. n = 3 experiments. Unpaired t-test.

Collectively, these results highlight Anisomycin, Geldanamycin, Bufalin and Gitoxigenin as promising structures for anti-fibrotic natural compounds in vitro.