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The antifibrotic effect of isolate tagitinin C from tithonia diversifolia (Hemsley) A. Gray on keloid fibroblast cell

Introduction Keloids characterized by fibroblast hyperproliferation and depositions of collagen which similar to cancer cells. Tagitinin C is a class of sesquiterpene lactones (SLS) was isolated from the leaves of the moon flower Tithonia diversifolia (Hemsley) A. Gray. The study aim is to evaluate the effects of tagitinin C from Tithonia diversifolia to keloid fibroblasts (KF). Methods Monolayer cultures of keloid fibroblast (three passages) were treated with 8 serial concentration of tagitinin C (0.015 to 2) μg/mL during 72 and 120 hours. A positive control using mitomycin C. Cellular viabilities were measured by MTT assay. Collagen depositions were measured by Sirius Red assay for nonsoluble collagen. Results The reading of the result was conducted by ELISA reader. Data were analyzed by probit regression with SPSS 19 for Windows. The result showed that tagitinin C can inhibit keloid fibroblasts (KF) viability with IC50 0.122 μg/mL (incubation 72h) and 0.039 μg/mL (120h), whereas mitomycin C IC50 0.120 μg/mL (72h) and IC50 of 0.100 μg/mL (120h). At IC50 concentration of tagitinin C on keloid collagen deposition 53.1% (72h) and 44.3% (120h), whereas the IC50 concentration of mitomycin C on keloid collagen deposition 60.4% (72h) and 52.1% (120h). Selectivity index tagitinin C on normal fibroblasts (NF) is 287 for 72h incubation and 791 for 120h incubation. Conclusion It can be concluded that the ability of tagitinin C inhibits KF viability and decreasing keloid collagen deposition is consistent with the concentration (concentration-dependent) and incubation time (time-dependent). Tagitinin C has a low toxicity level on NF with high selectivity index.


Introduction
Keloids are fibroproliferative benign tumors on the dermis layer of skin. It is caused by an excessive wound healing response [1]. In developing countries, there are around 100 million patients who have scarring complaints. Those are giving the impact on physical condition, aesthetic, psychological, and social [2,3]. Therefore, the research to looking for an antifibrotic agent keloids need to be developed. Keloids characterized by fibroblast hyper proliferations and depositions of collagen from fibroblast. The characteristic of fibroblast keloid is similar to the cancer cell characteristic [4]. Some of them are able to provide for their own growth signals, able to synthesize growth factor, able to replicate continuously, able to initiate angiogenesis signaling, and loss of ability to apoptosis. The difference is keloid fibroblasts unable to metastasis like cancer cell [5]. Tagitinin C is the class of sesquiterpene lactones (SLs) compounds, which is isolated from (Tithonia diversifolia(Hemsley) A.
Gray) leaf using Bioassay-Guided Isolation methods (MTT pada sel HeLa IC50: 9,776µg/mL) [6]. The previous study has done the cytotoxic test from pure isolate of Tithonia diversifolia against various human cancer cells and normal cells in vitro. It was done to determine its selectivity [7]. The previous research has shown that tagitinin C from Tithonia diversifolia tagitinin is the most active and the most selective in colon cancer cells (WiDr) with the selectivity index around 69.015. In addition, melanoma cells or skin cancer cells (M19) have the selectivity index around 40.536 (IC50 = 0,996μg/mL) [7,8]. The research on the antifibrotic effects of isolates tagitinin C from Tithonia diversifolia against keloid fibroblast cell has never been studied previously. Therefore, this research is conducted to examine the antifibrotic effects of isolates tagitinin C from Tithonia diversifolia against keloid fibroblasts which is measured from cell proliferation, deposition of collagen keloids, and toxicity tests of tagitinin C toward normal fibroblasts.

Methods
The subculture of keloids fibroblast: Subculturing was done by discarding medium in the flash. It was washed by 10 cc sterile PBS (Phosphate Buffer Saline). Then, fibroblasts harvested using warm trypsinization techniques. Trypsinization conducted by using trypsin 0.25% 2 cc and incubated for 2-3 minutes until all fibroblasts regardless from the basis flask. Trypsin was neutralized by adding complete DMEM medium until 3 times the volume of trypsin . The   cell suspension in the flask was transferred into 15 cc tube using a   Pasteur pipette, it was conducted by vortex then centrifuged with   the a speed of 200 MG for 10 min, the supernatant was discarded   carefully, cell was washed by 8 cc Sodium Chloride and it was   conducted by vortex, centrifuged with the 200g for 10 minutes and the supernatant was discarded carefully. The pellet was added with 1 cc of complete DMEM medium and conducted by vortex in order pellets and medium were mixed equitably. The mixture was divided into several sterile flasks and cultured in an incubator with 5% CO2 concentration, temperature 37°C, for 24 hours. The medium was changed every 3 days, until the cells fulfilling 50% on surface area flask, and can be harvested. The whole process of making the subculture could be repeated to obtain a subculture passage 3, after that the cells were harvested in the same way.

Results
Fibroblasts cell proliferation: A decreased amount of KF cell viability was found after the administration of tagitinin C isolate. The decrease in KF cell viability was in accordance with the concentration increase in tagitinin C isolates from Tithonia diversifolia with a strong correlation value (r = -0.924; p = 0.000 for 72 hours incubation; r = -0.919; p = 0.000 for 120 hours incubation) (Figure 1). Antifibrotic activity of tagitinin C isolates from Tithonia diversifolia and mitomycin C on KF cell proliferation was expressed in IC50 values. From the data above, the IC50 values were as follows. Results of independent t-test showed that IC50 value of tagitinin C between 72 hours and 120 hours incubations was significantly different (p < 0.05), which means that the longer the incubation time, the lower concentration of tagitinin C was needed to inhibit cell proliferation by 50% on KF cells population by in vitro, whereas the IC50 value of mitomycin C between 72 hours and 120 hours incubation was not significantly different (Table 1).

Antifibrotic effect of tagitinin C isolates from T. diversifolia on keloid collagen deposition
Result of keloid collagen deposition on tagitinin C IC50 concentration were 0.122 mg/mL (72 hours) and 0.039 mg/mL (120 hours), and mitomycin C IC50 were 0.120 mg/mL (72 hours) and 0.100 mg/mL (120 hours), presented in Table 2 as follows. Independent t-test results showed that keloid collagen deposition of tagitinin C and mitomycin C between 72 hours and 120 hours incubations was significantly different (p < 0.05), which means that the longer the incubation time, the fewer keloid collagen deposition, but the keloid collagen deposition that was given tagitinin C treatment was fewer than mitomycin C ( Table 2).  (Table 3).

Discussion
Cell proliferation is a physiological process of cell growth that is followed by cell division. Cell proliferation ability is balanced with the programmed cell death ability (apoptosis) in normal circumstances, resulting in a balance in the maintenance of tissues and organs integrity (homeostasis) [8]. On keloid, the regulatory function failed, resulting in an increase of fibroblast cell proliferation and a decrease in cell mortality [9]. From various studies that evaluated the effect of tagitinin C anticancer isolates, it can be presumed that tagitinin C isolates have the cytotoxic ability through various channels, for example, tagitinin C isolates can increase the expression of p53 protein in HeLa cells, thereby, can enhance the apoptosis ability [10]. Another study stated that tagitinin C isolates can reduce VEGF expression in colon cancer cells (WiDr) [11]. The VEGF expression inhibition mechanism by tagitinin C isolates is suspected to be caused by NF-kB transcription barriers through the alkylation on cystine residue (Cys 38) p56 [12,13]. This study also reported that that the longer the incubation time, the fewer keloid collagen deposition, but the keloid collagen deposition with tagitinin C treatment was fewer than treatment with mitomycin C. The collagen synthesis process began when the fibroblast got a stimulus from TGF-β that was bound to TGF-β receptors on the fibroblasts membrane. TGF-β normally stored in an inactive state and bound to a particular latent protein. In time of injury, the TGF-β will be separated from the binding protein and will be activated to respond to mechanical trauma that occurs [14]. In normal circumstances, the TGF-β signaling is regulated by peroxisome proliferatoractivated receptor gamma (PPAR-γ), which is one of the transcription factors on nuclear receptor that inhibits the fibrogenic excessive response [15]. Class of sesquiterpene lactones from T. diversifolia is known as PPAR α/γ dual agonist that includes tirotundin and tagitinin A [16]. Further research to examine the inhibition mechanism of keloid collagen deposition by tagitinin C isolates from T. diversifolia through PPARs is very interesting to be done.
Based on the toxicity test, tagitinin C of T. diversifolia increased the NF cell viability. Previous research showed that basal level of NF-κB in keloid fibroblast is higher than normal fibroblast. It explained that NF-κB also plays a role in the keloid pathogenesis, especially in the collagen synthesis of keloid [17]. Research on Xanthium stramarium (XAS) and Psoralea corylifolia (PSC), that is a compound of sesquiterpene lactones, is known to inhibit the keloid fibroblasts proliferation, inhibit TGF-β1, and inhibit collagen synthesis of keloid through the NF-κB [18]. Further research to look at the inhibition mechanism of keloid collagen deposition by tagitinin C isolates from T. diversifolia through inhibition of NF-κB is also very interesting to be done. The compound that is preferred for the development of new drugs is a selective compound. The means of selectivity is the selectivity in inhibiting the biological process that is only related to abnormal cell/tissue, without affecting normal cell/tissue. In this research, results showed that tagitinin C isolates from T. diversifolia has high selectivity to normal fibroblasts.

Conclusion
In the 72 hours incubation, IC 50 value of tagitinin C was 0.122 mg/mL with a keloid collagen deposition was at 53.1%, while in the 120 hours incubation, IC50value of tagitinin C was 0.039 mg/mL with keloid collagen deposition was at 44.3%. Based on the Sjogren et al. (2000) criteria, tagitinin C of T. diversifolia was classified into low toxicity category against the NF cells with a high index of selectivity.
In summary, tagitinin C has a potential activity to inhibit keloid fibroblast viability and to decrease keloid collagen deposition. This study proved a novel evidence that tagitinin C is non-toxic and selective for anti-fibrotic agent.
What is known about this topic  Fibroblast hyperproliferation and depositions of collagen;  Tagitinin C's effects to Keloid Fibroblast.  Tagitinin C has a low toxicity level on NF with high selectivity index.     Mitomycin C 0,120 ± 0,034 0,100 ± 0,051 0,621   Page number not for citation purposes 8 Figure 2: Correlation between concentration of tagitinin C isolates from T. diversifolia on the cells viability of normal fibroblast with 72 hours and 120 hours incubation times