Antitumor Activity of Ethyl Acetate Extraction of Wikstroemia chamaedaphne: Cell Cycle Arrest and Apoptosis- Inducing Activity in Melanoma Cells

The ethyl acetate extraction (WCE) of Wikstroemia chamaedaphne Meisn on the viability of melanoma cell lines B16 and A375 were evaluated. The WCE exhibited a potent cytotoxic against B16 and A375 with IC50 values of 156.2 and 192.8 μg/mL, respectively. Cell migration was assessed using a wound healing assay. The WCE caused cell cycle arrest in the G0/G1 phase in B16 cells and the S phase in A375 cells, according to cell cycle analysis. Flow cytometric analysis revealed that WCE promoted apoptosis in B16 and A375 cells in a dosedependent manner. Chemical analysis of WCE resulted in the isolation of a new diterpenoid, wikstchalin A (6) with five known flavonoids (1−5) and two known diterpenoids (7 and 8). Spectroscopic analysis revealed their structures, and CD analysis revealed the absolute configurations of 6. The absolute structure of pimelotide A (7) was firstly confirmed by single-crystal X-ray diffraction. The cytotoxicities of all the compounds (1−8) were evaluated against B16 cell lines. Compound 8 was more active than the positive control, dacarbazine (IC50 300 μM), in terms of cytotoxicity against B16, with an IC50 value of 7.9 μM.


Introduction
Malignant melanoma is one of the most common malignancies. Around 232100 cases of all newly diagnosed primary malignant cancers are cases of cutaneous melanoma, and there are 55500 newly deaths worldwide every year [1]. Malignant melanoma is frequently resistant to chemotherapy and radiotherapy, and advanced melanoma patients have a dismal prognosis, with a median overall survival of 8-10 months and a 5-year survival rate of 6% [2]. Metastatic melanoma is one of the most difficult tumors to treat, imposing a financial burden on both individual patients and society. Despite major breakthroughs in chemotherapy regimens, there has been little change in malignant melanoma mortality during the last several decades. The finding of exact molecular targets for malignant melanoma chemoprevention is believed to be an accessible option for the development of new preventive medicines.
Wikstroemia chamaedaphne Meisn., a toxic shrub native to China, has been used in traditional Chinese medicine to treat edema, cough, hepatitis, schizophrenia, and infertility [3]. Previous chemical investigations on the W. chamaedaphne have resulted the isolation of daphnane diterpenoids [4][5][6], flavonoids [5], and phenolic constituents [7]. As part of our screening program to investigate antineoplastic compounds from this genus [8], ethyl acetate extraction (WCE) of W. chamaedaphne demonstrated cytotoxic activity. The WCE was tested for cytotoxicity against two melanoma cell lines, B16 and A375, respectively. The WCE was found to be moderate activity to the melanoma cell lines B16 and A375, with IC50 values of 156.2 and 192.8 μg/mL, respectively. Chemical investigation of WCE has led to the isolation of a new wikstchalin A (6) with the five known flavonoids (1−5) and two known diterpenoids (7 and 8). Spectroscopic analysis revealed their structures, and CD analysis revealed the absolute configurations of 6. The absolute structures of pimelotide A (7) were firstly confirmed by single-crystal X-ray diffraction. Herein, details of cytotoxic activities of WCE, the isolation, and structural elucidation are described.

General Methods
At 25°C, NMR spectra were collected using a Bruker AM-600 spectrometer. A semi-preparative HPLC Eclipse XDB-C18 column was used in conjunction with an Agilent 1200 series HPLC system. For column chromatography (CC), we used a silica gel (300-400 mesh, Qingdao Haiyang Chemical Co., Ltd.), a C18 reversed-phase silica gel (12 nm, S-50 μm, YMC Co., Ltd.), and a Sephadex LH-20 gel (Amersham Biosciences). All of the solvents were analytical or chromatographic grade (Shanghai J&K Scientific Ltd or Chemical Reagents Company, Ltd.).

Plant Material
W. chamaedaphne flower buds were purchased from Bozhou Jinwan Pharmaceutical Company (Bozhou, People's Republic of China) and collected in November 2019 from Taiyuan, Shanxi Province, People's Republic of China. One of the authors (L. Luo) identified the specimen. A standard sample (SWCZ-201911) was deposited at the Central Laboratory, Seventh People's Hospital of Shanghai University of TCM.

Cell Culture
B16 mouse malignant melanoma and A375 human malignant melanoma cells were provided by the cell bank of the Chinese Academy of Sciences (Shanghai, China). B16 cells were thawed and grown in RPMI-1640 and supplemented with 0.5% penicillin/streptomycin and 10% FBS, while A375 cells were thawed and expanded in DMEM. The cells were incubated at 37 °C with 5% CO2. For the following assays, all cells were harvested during the exponential growth phase.

Cell Viability Assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell viability. Separately, B16 and A375 cells were seeded into 96-well plates at a density of 5×10 3 cells/well and incubated overnight. Following a 24-hour incubation with WCE extract at various concentrations, B16 and A375 cells were treated with MTT (5 mg/mL dissolved in PBS) for an additional 4-hour incubation at 37 °C. Following the dissolution of the formazan crystals in DMSO, the absorbance at 560 nm was measured with a spectrophotometer and the IC50 was calculated using GraphPad 8. All assays were carried out in triplicate.

Morphological Analysis
The AO/PI staining assay was used to investigate the morphological changes that occurred in the cells after WCE treatment. For 24 hours, cells were treated with 100, 200, 300, and 400 μg/mL of WCE, respectively. 300 μM of dacarbazine (DBZ) were used as positive controls. Acridine orange (AO) and propidium iodide were used to stain B16 and A375 cells (PI). The plate was incubated in the dark for 5 minutes before being washed with PBS to remove the unbound dye. An EVOS FL digital microscope (200 objective) was used to capture the cell morphology. All assays were carried out in triplicate.

Wound Healing Assay
B16 and A375 cells (1 × 10 6 cells/well) were seeded and incubated until the plates were full. To assess cell migration, wound lines in the shape of a cross were made in confluent cells by scraping with a plastic 200 μL pipette tip. After wounding, floating cells were washed with PBS and cultured for 48 hours in 2 percent FBS-containing DMEM or RPMI-1640 supplemented with or without WCE. Data were collected as previously described [9]. Specifically, under a microscope, the width of the wound healing was then observed and photographed. All assays were carried out in triplicate.

Cell Cycle Analysis
B16 and A375 cells were harvested and washed twice with cold PBS after 24 hours of WCE or DBZ treatment. The cells were fixed overnight in 75% frozen ethanol at 4°C before being resuspended in 1 mL cold PBS. After adding 500 μL of dye solution, the cells were incubated in the dark at room temperature for 30 minutes before being detected by flow cytometry. All assays were carried out in triplicate.

Cell Apoptosis Analysis
The effect of WCE on cell apoptosis was investigated using Annexin V-FITC apoptosis detection in conjunction with flow cytometry. For 24 hours, B16 and A375 cells were treated with varying doses of WCE or DBZ. After washing with PBS, cells at a concentration of 5 × 10 4 cells/mL were gently mixed with 5 μL Annexin V-FITC and 5 μL PI. Cells were then left at room temperature in the dark for 15 minutes. The cells were then placed in 400 μL binding buffer before being loaded onto the FACS Calibur. FLOW JO 10 was used for additional analysis. As controls, cells were left untreated or stained with Annexin V-FITC or PI alone. All assays were carried out in triplicate.

Cytotoxic Effects of WCE Against Melanoma Cells
An MTT assay was used to assess the WCE's in vitro cytotoxicity against B16 and A375 melanoma cell lines in vitro. WCE was found to have significant cytotoxic effects against B16 and A375, with IC50 values of 156.2 and 192.8 μg/mL after 24 hours of treatment, respectively ( Figure 1).

Morphological Changes in Melanoma Cells Treated with WCE
To confirm the apoptosis-inducing effects of WCE, melanoma cells were examined in fluorescence photomicrographs of B16 and A375 cells stained with acridine orange (AO) after treatment with various concentrations of WCE for 24 hours. After that, the inverted light or fluorescence microscope was used to examine the morphological features of apoptosis. The cells in the control group had normal morphology and were stained green in the nuclei. When the WCE concentration was between 100 and 200 µg/mL, the cell membrane shrank and the nuclear chromatin started to pyknosis, indicating the early manifestation of apoptosis. Most of the nuclei were yellow green and bead-like when the WCE concentration was 300 µg/mL, and the nuclear chromatin was orange red and pyknosis, indicating that many of the cells had entered the late stage of apoptosis. When the WCE concentration was raised to 400 µg/mL, the cell membrane ruptured and the nucleus turned red (Figure 2). Thus, WCE could cause apoptosis in B16 and A375 cells in a dose-dependent manner.

Antimigratory Activity of the WCE in Melanoma Cells
Wound-healing assays were used to investigate the effects of WCE on melanoma cell migration. B16 and A375 cells were treated with different concentrations of WCE for a total of 48 hours to see how it affected migration. The wound was measured in width after incubation and compared to the original ones. WCE's anti-migration activity was assessed using this method of analysis. In B16 and A375 cells, the WCE has equivalent activity to the positive control DBZ (Figure 3).

The WCE Induce Cell Cycle Arrest in B16 and A375 Melanoma Cells
The cell cycle profiles of B16 and A375 cells were analyzed using flow cytometry to further investigate the effects of WCE on the cell cycle. The cells were stained with PI after being treated with WCE at concentrations of 100, 200, 300, and 400 µg/mL for 48 hours. The cells were then flow cytometrically examined to determine their DNA content. When the concentration of WCE was increased, the percentage of cells in G0/G1 phase increased from 58.94% to 59.45%, 62.37%, 69.41 %, and 80.41 %, respectively, while the number of cells in S and G2/M phase decreased gradually ( Figure  4A), indicating that WCE could block the cell cycle in G0/G1 phase to inhibit B16 cell proliferation. The percentage of cells in the S phase increased significantly after WCE treatment, while the percentage of cells in the G0/G1 and G2/M phases decreased significantly ( Figure 4B). S phase values increased from 23.14% to 31.17%, 43.05%, 45.17%, and 45.63%, respectively, when compared to the control group, indicating that WCE could block the cell cycle in S phase to inhibit the proliferation of A375 cells.

The WCE Induce Apoptosis in B16 and A375 Melanoma Cells
The percentage of apoptotic cells was determined using flow cytometry and Annexin V-FITC/PI double staining. As shown in Figure 5, cells considered viable were annexin V-FITC and PI negative, cells in early apoptosis were annexin V-FITC positive and PI negative, and cells in late apoptosis or already dead were both annexin V-FITC and PI positive. The percentages of total apoptotic cells in B16 were 27.35 %, 45.38%, 69.18%, and 81.03% after 24 hours of treatment with 100, 200, 300, and 400 μg/mL, respectively, and the percentages of total apoptotic cells in A375 were 36.54%, 53.26%, 70.59%, and 74.81 %. These findings revealed that WCE induced apoptosis in B16 and A375 cells was dosedependent.

Cytotoxic Effects of the Isolates
The MTT assay was used to assess the in vitro cytotoxicity of all isolated compounds 1−8 against B16 cell lines. Compound 8 was discovered to be the most active compound, with an IC50 value of 7.9 μM, compared to the positive control DBZ against B16, which had an IC50 value of 300 μM.

Conclusion
The WCE was found to be highly cytotoxic to the melanoma cell lines B16 and A375, with IC50 values of 156.2 and 192.8 µg/mL, respectively. In an acridine orange and propidium iodide (AO/PI) staining assay, WCE-treated B16 and A375 cells showed typical morphological changes of cell apoptosis. To assess cell migration, a wound healing assay was used. WCE induced cell cycle arrest at the G0/G1 phase in B16 cells and the S phase in A375 cells, according to cell cycle analysis with propidium iodide staining. WCE induced apoptotic events in B16 and A375 cells in a dose-dependent manner, according to flow cytometric analysis using Annexin V-FITC and propidium iodide staining. These findings strongly suggested that WCE could be developed further as a potential antitumor agent. Chemical analysis of the WCE resulted in the isolation of a new wikstchalin A (6), in addition to the five known flavonoids (1−5) and two known diterpenoids (7 and 8). Single-crystal X-ray diffraction was used to confirm one of the diterpenoids, pimelotide A (7). All compounds were tested for cytotoxicity against the B16 cell line, and compound 8 inhibited B16 with an IC50 values of 7.9 μM, making it more active than the positive control, DBZ (IC50 = 300 μM). However, the mechanism of compound 8's inhibition of B16 cell line proliferation requires further investigation.