In Vitro Wound Healing and Anticancer Effects of Ixora coccinea in Malignant Melanoma Cell Lines

Abstract

Background Ixora coccinea is a medicinal plant with many active constituents that are responsible for wound healing and have anticancer properties. Herbal extracts increase the mechanisms related to wound healing, like blood clotting, fighting infection, and epithelialization. The effect responsible for this property may be the presence of phytoconstituents like flavonoids, polyphenols, and alkaloids. Many researchers have evaluated the wound-healing effect of I. coccinea leaf extract in aqueous methanol. This study aimed to determine the in vitro wound healing and anticancer efficacy of I. coccinea leaf ethyl acetate extract and evaluate the in silico docking of the selected phytoconstituents of I. coccinea in the 2vcj protein. Materials and methods The human dermal fibroblast cell line was used to determine the rates of cell migration and proliferation for evaluating the wound-healing effect of the I. coccinea leaf ethyl acetate fraction. 4',6-diamidino-2-phenylindole (DAPI) fluorescence labeling was used to estimate the rate of cell migration. The one-step TUNEL (TdT-mediated dUTP Nick-End Labeling) in situ apoptosis kit and the annexin V-FITC/7-AAD apoptosis kit were used to perform DNA damage assays in the malignant melanoma cell line. The ethyl acetate fraction of I. coccinea leaves was analyzed for its impact on wound healing markers, including keratin-10, keratin-14, type IV collagen, and α-SMA. Results The wound-healing nature was interesting in the ethyl acetate fraction at doses of 50 µg/mL and 100 µg/mL. Both studies involved in the DNA damage study against malignant melanoma cell lines showed the cleavage of apoptotic cancer cells, which was detected using a fluorescence microscope. When compared with the control, a dose of 100 μg/ml of ethyl acetate fraction from the leaves of I. coccinea showed fibroblast migration of cells into the wound area. The statistical values were considered significant at the level of P < 0.05. An in silico docking study on the 2vcj protein revealed that selected phytoconstituents of I. coccinea resulted in good docking scores to inhibit Hsp90. Conclusion I. coccinea ethyl acetate leaf extract can inhibit the growth of malignant melanoma cell lines and promote wound healing, as shown by the study results. It might be a viable therapeutic modality for skin cancer.

Keywords: 2vcj protein; apoptosis; dna damage; fluorescence microscope; human dermal fibroblasts.

Figure 1. Photomicrograph representation of the in vitro scratch assay of Ixora coccinea leaf ethyl acetate extract at different concentrations at different time intervals.

A: Control at 0 hour; B: magnified view of control at 24 hours; C: magnified view of control at 48 hours, D: Ixora coccinea leaf ethyl acetate extract 50 µg/mL at 24 hours; E: Ixora coccinea leaf ethyl acetate extract 50 µg/mL at 48 hours; F: Ixora coccinea leaf ethyl acetate extract 100 µg/mL at 24 hours; G: Ixora coccinea leaf ethyl acetate extract 100 µg/mL at 24 hours; H and I: magnified view of 100 µg/mL at 48 hours.

Figure 2. Wound closure (mm) by Ixora coccinea leaf ethyl acetate extract at 100 µg/mL at different time intervals.

Figure 3. Expression of wound healing markers in human dermal fibroblast cell lines by Ixora coccinea leaf ethyl acetate extract at 100 µg/mL.

A: Keratin 10 marker; B: Keratin 14 marker; C: Type IV collagen marker; D: α- SMA marker.

Figure 4. Apoptosis detected by one-step TUNEL in situ apoptosis kit in malignant melanoma cell lines using Ixora coccinea leaf ethyl acetate extract at different concentrations.

A: control; B: Ixora coccinea leaf ethyl acetate extract 50 µg/mL; C: Ixora coccinea leaf ethyl acetate extract 100 µg/mL.

Figure 5. Apoptosis detected by Annexin V-FFITC/7-AAD in malignant melanoma cell lines using Ixora coccinea leaf ethyl acetate extract at different concentrations.

A: control; B: Ixora coccinea leaf ethyl acetate extract 50 µg/mL; C: Ixora coccinea leaf ethyl acetate extract 100 µg/mL.

Figure 6. Two-dimensional interactions of camptothecin with the 2vcj protein.

Figure 7. Two-dimensional interactions of quercetin with the 2vcj protein.

Figure 8. Two-dimensional interactions of rutin with the 2vcj protein.

Figure 9. Three-dimensional interactions of camptothecin, quercetin, and rutin with the 2vcj protein.