The effects of patchouli alcohol and combination with cisplatin on proliferation, apoptosis and migration in B16F10 melanoma cells

Abstract Melanoma is a highly metastatic cancer with a low incidence rate, but a high mortality rate. Patchouli alcohol (PA), a tricyclic sesquiterpene, is considered the main active component in Pogostemon cablin Benth, which improves wound healing and has anti‐tumorigenic activity. However, the pharmacological action of PA on anti‐melanoma remains unclear. Thus, the present study aimed to investigate the role of PA in the proliferation, cell cycle, apoptosis and migration of melanoma cells. These results indicated that PA selectively inhibited the proliferation of B16F10 cells in a dose‐ and time‐dependent manner. It induced cell cycle arrest at the G0/G1 phase and typical morphological changes in apoptosis, such as chromatin condensation, DNA fragmentation and apoptotic bodies. In addition, PA reduced the migratory ability of B16F10 cells by upregulating E‐cadherin and downregulating p‐Smad2/3, vimentin, MMP‐2 and MMP‐9 expression. PA was also found to strongly suppress tumour growth in vivo. Furthermore, PA combined with cisplatin synergistically inhibited colony formation and migration of B16F10 cells and attenuated the development of resistance to treatment. Therefore, the results of this study indicate that PA may play a pivotal role in inducing apoptosis and reducing the migration of melanoma cells, and may thus be a potential candidate for melanoma treatment.

dicated that PA selectively inhibited the proliferation of B16F10 cells in a dose-and time-dependent manner. It induced cell cycle arrest at the G 0 /G 1 phase and typical morphological changes in apoptosis, such as chromatin condensation, DNA fragmentation and apoptotic bodies. In addition, PA reduced the migratory ability of B16F10 cells by upregulating E-cadherin and downregulating p-Smad2/3, vimentin, MMP-2 and MMP-9 expression. PA was also found to strongly suppress tumour growth in vivo. Furthermore, PA combined with cisplatin synergistically inhibited colony formation and migration of B16F10 cells and attenuated the development of resistance to treatment. Therefore, the results of this study indicate that PA may play a pivotal role in inducing apoptosis and reducing the migration of melanoma cells, and may thus be a potential candidate for melanoma treatment.

| BACKG ROU N D
The latest report from the World Health Organization in 2018 showed that there were 288,000 new cases and 61,000 deaths associated with melanoma. 1 Melanoma is a highly malignant cancer with a high mortality rate mainly because of its high metastatic ability and resistance to various treatments. 2 When patients are diagnosed with melanoma, most have advanced to middle or advanced stages, with obvious symptoms of metastasis. Once the cancerous cells spread to other tissues or organs of the body, they cannot be cured by surgery, which is related to more than 90% of deaths by melanoma. 3 Cytotoxic chemotherapy, including a combination of cisplatin (CDDP) and doxorubicin (DOXO), has been widely used as the main treatment for melanoma. 4 However, it induces apoptosis in both melanoma and normal tissues and exhibits undesirable side effects, such as bone marrow toxicity, nephrotoxicity, hepatotoxicity and gastrointestinal toxicity. 5,6 Although chemotherapy suppresses the growth of tumours, this effect is not long-lasting in most cases, resulting in drug resistance in melanoma, leading to treatment failure. 7 Together, the clinical benefits of these approaches are affected by the cytotoxicity, drug resistance and drug selection of cells. Hence, it is important to determine how to prolong the effective duration of drugs and reduce side effects to improve the quality of life of patients. Conventional drugs are insufficient to improve the status of treatment, and it is necessary to develop new chemotherapeutic drugs to overcome the current limitations of melanoma therapy.
The demand for the development of novel anticancer drugs has been directed towards research on potentially useful natural products in herbs, vegetables and fruits. 8,9 Approximately 40% of approved commercial drugs and 64.9% of existing anti-cancer drugs are derived from natural products or their derivatives. 10 They are an important source of compounds with biological activities against various diseases and have low toxicity and side effects. 11 In addition, there are several studies that used herbal medicine to treat malignant melanoma, which indicate anti-proliferative, pro-apoptotic and anti-metastatic effects. For example, hispidulin, a flavone distributed in plants of the Asteraceae, reduced cell growth by decreasing AKT and ERK phosphorylation, inducing apoptosis via activation of caspases and inhibiting cell migration through downregulation of matrix metalloproteinase-2 expression in A2058 human melanoma cells. 12 Theaflavin, a primary pigment of tea, suppressed cell proliferation, induced early and late apoptosis and inhibited migration/invasion through the regulation of relative mRNA and protein expression in B16F10 mouse melanoma cells. 13 Pogostemon cablin Benth. is a medicinal plant that is commonly used to treat fever, headache, nausea, diarrhoea and facial diseases and is widely cultivated in the Philippines, Malaysia, India and China. [14][15][16] The extracts of P. cablin are one of the most important ingredients in cosmetics and exhibit soothing and anti-dermatophytic, antioxidative and anti-inflammatory effects on the skin. [16][17][18] Patchouli alcohol (PA), a tricyclic sesquiterpene, is considered a major effective component of P. cablin and is used in the quality control of patchouli oil in the pharmaceutical industry. Several studies have demonstrated that PA has multiple effects, such as anti-oxidation, 19 antiinflammatory, 20 anti-influenza virus, 21 prevention of UV-induced skin photoaging, 22 improvement of wound healing, 23 protection against myocardial ischaemia-reperfusion injury 24 and anti-tumorigenic activity in colorectal, lung and prostate cancer. [25][26][27] However, despite these diverse beneficial effects, studies on the use of PA in the treatment of melanoma are lacking. The present study aimed to investigate the anticancer effects of PA on melanoma cells. The current data revealed that PA suppresses the growth of B16F10 cells both in vitro and in vivo by regulating proliferation, apoptosis and migration. These findings indicate that PA may be a potential compound for the treatment of melanoma and provide more insights for the development of novel agents or functional foods from medicinal plants.

| Chemicals and reagents
Patchouli alcohol and cisplatin were purchased from Wuhan ChemFaces Biochemical Co., Ltd. and the Cayman Chemical Company, respectively. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet razolium bromide (MTT), dimethyl sulfoxide (DMSO) and propidium iodide (PI) were purchased from Sigma-Aldrich and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay kit was purchased from Roche. Supersensitive link-label immunohistochemistry (IHC) detection system and 3,3′-diaminobenzidine (DAB) were purchased from BioGenex. For western blotting and IHC staining experiments, primary antibodies against proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4), caspase-8, caspase-9, caspase-3, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9 were purchased from Santa Cruz Biotechnology; Smad2/3, p-Smad2/3 were purchased from Cell Signalling Technology; E-cadherin, vimentin and β-actin were purchased from iReal Biotechnology Co., Ltd. In vitro experiments, PA (purity ≥98%) was prepared as a 224.9 mM stock solution in DMSO and stored at −20°C. The stock solution was diluted with the complete medium for subsequent applications, containing <1% final concentration of DMSO. In vivo experiments, the powder of PA was dissolved in mineral oil and prepared as 75 and 150 mg/kg for subcutaneous injection. An equal volume of DMSO or K E Y W O R D S cell apoptosis, drug combination, melanoma, migration, patchouli alcohol mineral oil was used as a negative control in vitro and in vivo experiments, respectively.

| Cell culture
The murine B16F10 melanoma and NIH/3T3 fibroblast cell lines were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), HEPES buffer solution, sodium pyruvate and penicillin/streptomycin at 37°C in a humidified 5% CO 2 incubator. All the reagents used for cell culture were purchased from Gibco.

| Cell viability assay
The inhibitory effect of PA on B16F10 cells was evaluated via cell viability using the MTT assay. The cells were seeded into 96-well plates at 5 × 10 3 cells/well in 100 μL medium for 24 h adherence (40%-50%

| Cell cycle analysis
The cell cycle distribution in PA-treated cells was determined by PI staining. Cells were treated with 89.9 μM PA for 0, 6, 12, 24 and 48 h.
Then, the treated cells were harvested and incubated with the solution of PI (40 μg/mL) and RNase A (100 μg/mL) overnight at 4°C.
The cell cycle distribution was detected by FACScan flow cytometry (Beckton Dickinson) and calculated using FlowJo software (Treestar).

| Apoptosis detection
DNA cleavage was assessed using the TUNEL assay kit following the manufacturer's instructions. B16F10 cells were plated in 10 cm dishes (2 × 10 6 cells/dish), cultured for 24 h and then treated with 89.9 μM PA. After further 6-48 h, the PA-treated cells were fixed with 10% neutral buffered formalin for 10 min and stored at 4°C.
Treated cells or tumour tissues were incubated with 3% H 2 O 2 in methanol, permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate on ice and incubated with TUNEL solution for 1 h at 37°C. PI staining (red fluorescence) was used as a contrast dye to highlight the nuclei. TUNEL assay results (green fluorescence) were observed using a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Thornwood, NY, USA). Finally, the membranes were exposed to enhanced chemiluminescence reagent (ECL, T-Pro Biotechnology) and detected using a fluorescence/chemiluminescence imaging analyser (GE LAS-4000, GE healthcare Life Sciences). The ImageJ 1.47t software was used for densitometry analysis and normalized against β-actin expression.

| In vivo experiments in mice
The anti-tumour activity of PA in vivo was studied according to the prescribed guidelines for the Care and Use of Laboratory Animals. week. Mice were sacrificed using carbon dioxide when the tumour volume exceeded 1500 mm 3 . Tumour tissues were fixed in 10% formalin, embedded in paraffin and sliced, followed by staining with TUNEL, haematoxylin and eosin (H&E) and IHC (caspase-3, PCNA, VEGF, MMP-2 and MMP-9) for further histological examination. 28

| Colony formation assay
A plate clonogenic assay was used to test the colony formation ability of B16F10 cells. The cells were digested and counted, and approximately 2000 cells were seeded into a 24-well plate and incubated for 24 h. The cells were treated with 45.0 μM PA and/or 6.6 μM cisplatin in triplicate for 24 h, followed by culturing in a drug-free medium with 5% FBS for another 7 days. Subsequently, the cells were washed with PBS, fixed with 100% methanol and stained with 0.1% crystal violet and then colonies (>50 cells) in each well were counted using a light microscope.

| Migration assay
The Boyden chamber assay was used to detect the migration of melanoma cells. Briefly, B16F10 cells were starved for 24 h, 1.0 × 10 5 cells suspended in 100 μL serum-free medium were added to the upper chamber, and 100 μL culture medium (10% FBS) containing 67.5 μM PA and/or 6.6 μM cisplatin was added to the lower chamber.
After incubation at 37° for 24 h, the cells in the upper chambers were removed, then the migrated cells were fixed with 100% methanol and stained with 0.1% crystal violet for 10 min each. Images of migrated cells in five randomly selected fields were captured, and cell numbers were calculated under a light microscope.

| In vitro resistance assay
B16F10 cells were plated in 96-well plates and allowed to attach for 24 h. PA (89.9 μM) and/or cisplatin (6.6 μM) were added in three technical replicates for 5 and 10 days, and fresh media and drugs were added every 3 days for the duration of the experiment. The treated cells were fixed with 100% methanol and stained with 0.1% crystal violet. Finally, 50 μL of 10% acetic acid was added to each well and the OD was measured at 560 nm using a microplate reader. Cell survival (%) = (PA − treated OD/control OD on day 5) × 100.

| Statistical analysis
Data are presented as the mean ± SD (in vitro) or mean ± SEM (in vivo). The unpaired Student's t-test was used to compare two groups, one-way anova was used to compare multiple groups and the Kaplan-Meier method was used to perform survival analysis, using Excel 2016 software or SPSS v16.0 software. Statistical significance was set at p < 0.05.

| PA inhibited cell growth and induced cell cycle arrest in G 0 /G 1 phase
The chemical structure of PA with a molecular weight of 222.37 g/ mol is shown in Figure 1A.

| PA influenced the cell morphology and triggered apoptosis in melanoma cells
As shown in Figure 2A, the morphology of B16F10 cells exhibited significant changes after treatment with PA. The PA-treated cells began to shrink, turned circular, the cell edges became irregular and many dead cells were suspended in the medium. Besides, the percentage of cells at subG 1 phase was enhanced from 8.54% ± 0.56% to 8.32% ± 0.31%, 7.95% ± 1.27%, 16.54% ± 1.30% and 35.58 ± 3.18%, respectively, with increasing PA treatment time ( Figure 2B). To determine cells undergoing apoptosis, a TUNEL assay was performed to evaluate the proapoptotic effect of PA on B16F10 cells. Cells treated with 89.9 μM PA for 48 h showed many fragmented nuclei with green fluorescence and apoptotic morphology, such as chromatin condensation, DNA fragmentation and apoptotic bodies ( Figure 2C). The number of TUNEL-positive cells was enhanced with increasing PA treatment time, measured by cell counting (Figure 2D). These results demonstrated that PA exerted time-dependent pro-apoptotic effects on B16F10 cells. Results are presented as the mean ± SD of at least three independent experiments. *P < 0.05, versus control with a significant increase. # P < 0.05, versus control with significant decrease. cycle. Furthermore, the expression of pro-caspase-8, 9 and 3 was significantly reduced, while cleaved-caspase-8, 9 and 3 was significantly increased with treatment time, suggesting that extrinsic and intrinsic apoptosis pathways were activated by PA treatment in melanoma cells.

| PA attenuated the migratory abilities of B16F10 cells
The metastatic ability of tumour cells poses a major threat to cancer-  To study the underlying mechanism of PA in inhibiting melanoma, histological analysis was used to detect markers of apoptosis, proliferation, angiogenesis and metastasis in vivo. As shown in Figure 6  The treated cells were inhibited at day 5 and re-grew at day 10, indicating that the cells developed resistance to cisplatin; however, the combination of drugs reduced and delayed this effect ( Figure 7D).

| PA combined with cisplatin reduced colony formation, migration and drug resistance abilities
Taken together, these results demonstrate that PA enhances the antiproliferative and anti-migratory activity of cisplatin and attenuates the development of cisplatin resistance in melanoma cells.

| DISCUSS ION
Compounds from natural sources have been widely investigated for cancer therapy because of their availability and low toxicity compared to standard chemotherapeutic agents. 11 Pogostemon

F I G U R E 3
The expression levels of cell cycle and apoptosis relative proteins regulated by PA treatment. Cells were incubated with 89.9 μM of PA for 0, 6, 12, 24 and 48 h, and the expression level of proteins associated with cell cycle and apoptosis was detected using western blotting. PA treatment dramatically attenuated the expressions of PCNA, cyclin D1 and CDK4, while activating caspase-8, caspase-9 and caspase-3. Data are represented as means ± SD of at least three independent experiments. *P < 0.05, versus control with a significant increase. # P < 0.05, versus control with a significant decrease. C-Cas, cleaved-caspase; PCNA, proliferating cell nuclear antigen; Pro-Cas, pro-caspase. At present, an increasing number of researchers believe that inducing cell cycle arrest and apoptosis may be a new therapeutic strategy for cancer prevention and treatment. The cell cycle is strictly regulated by these genes. The key to carcinogenesis is uncontrolled cell growth and proliferation due to abnormal regulation of the cell cycle. 33 Based on changes in DNA content, the cell cycle is divided into four stages: G 1 , S, G2 and M phases. In the current study, the cell cycle was analysed using PI staining and flow cytometry to identify the effects of PA treatment on the cell cycle. The results revealed that the cell cycle of B16F10 cells exposed to PA was arrested in the G 0 /G 1 phase. A combination of cyclins, CDKs, and CDK inhibitors mediates the regulation of cell cycle distribution at critical restriction points. 34 Among them, cyclin D1 and CDK4 are important regulators that promote the transition from G 1 to S phase.
We detected the expression of proteins associated with cell cycle arrest through western blotting and found that the expression of cyclin D1 and CDK4 decreased in a time-dependent manner after PA treatment.
It is well known that the induction of cell cycle arrest can trigger apoptosis. Apoptosis is an essential mechanism that induces cell As a well-known chemotherapeutic drug, cisplatin has been widely used to treat a variety of solid cancers, including ovarian, lung and testicular cancer. 42 The most important anticancer mechanism of cisplatin is to trigger DNA damage and induce cell apoptosis, leading to the F I G U R E 6 PA regulated markers expression of proliferation, apoptosis, angiogenesis and metastasis in vivo. The mice (vehicle and 150 mg/kg PA groups) were sacrificed when the tumour volume exceeded 1500 mm 3 , and then the tumour was fixed, sliced and stained with H&E (scale bar: 100 μm), IHC (scale bar: 50 μm) and TUNEL (scale bar: 50 μm) for histological examination. Image of H&E staining showed the morphology of cell death and nucleolysis (arrow). The expression of caspase-3, PCNA, VEGF, MMP-2 and MMP-9 were examined using immunohistochemical analysis and scored using the Quickscore method. PA-induced cell apoptosis was determined using tissue TUNEL stain. All data are shown as mean values ± SEM. *P < 0.05, versus vehicle group. H&E, haematoxylin and eosin; IHC, immunohistochemistry; MMP, matrix metalloproteinases; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; VEGF, vascular endothelial growth factor. death of cancer cells. Cisplatin is beneficial for the overall survival and relapse-free survival of patients with advanced melanoma and metastatic melanoma. 43 However, single-agent chemotherapy has low response rates with a median duration of approximately 3 months and undesirable side effects such as ototoxicity, nephrotoxicity, hepatotoxicity and gastrointestinal toxicity. 42,44 To overcome this limitation, F I G U R E 7 Combination effects of PA combined with cisplatin on proliferation, colony formation, migration and drug-resistant abilities. (A) Cells were treated with indicated concentrations of PA and cisplatin for 48 h, and measured cell viability using MTT assay. The combination index (CI) was calculated by CompuSyn software, using to evaluate the synergism (CI < 1), additive effects (CI = 1) and antagonism (CI > 1). *P < 0.05, versus PA only. (B) B16F10 cells were treated with PA (45.0 μM) and/or cisplatin (6.6 μM) for 24 h and subsequently cultured in drug-free medium containing 5% FBS for another 7 days. The colonies were fixed using 100% methanol, stained with crystal violet and counted microscopically. (C) Boyden chamber assay of B16F10 cells upon treatment with PA (67.5 μM) and/or cisplatin (6.6 μM) for 24 h, after which the migrated cells were stained, photographed and quantified. (D) For in vitro resistance assay, after long-term culture with PA (89.9 μM) and/or cisplatin (6.6 μM) for 5 and 10 days, the treated cells were fixed, stained and dissolved in 10% acetic acid, followed by O.D. measurement at 560 nm using a microplate reader. Survival cells of the control group at day 5 were considered as 100%. Data are represented as means ± SD in three technical replicates. *P < 0.05, versus control. # P < 0.05, versus PA. & P < 0.05, versus cisplatin. $ P < 0.05, versus day 5. CP, cisplatin. combinations of cisplatin with other agents or natural products with different mechanisms of action have been widely reported. 42,45 The current study showed that PA combined with cisplatin exhibited synergistic inhibitory effects on the colony formation and migration of B16F10 cells. In addition, PA strongly reduced the regrowth of cisplatin-treated cells, indicating that PA attenuated the development of resistance to cisplatin. Taken together, PA may not only be a potential approach for melanoma treatment but also a sensitizer for cells resistant to cisplatin or other conventional chemotherapeutic drugs, suggesting that it has good clinical application prospects.
Because human melanoma is an aggressive cancer type, in vitro and in vitro experiments were performed in the current study using the highly aggressive murine melanoma cell line B16F10 to evaluate the inhibitory effect of PA on melanoma cells and animal models, respectively. B16 melanoma, a spontaneous melanoma derived from C57BL/6 mice, is the most commonly used model of metastatic melanoma for preclinical studies. The B16F10 cell line, the most frequently used variant, was generated as the 10th serial passage subclone of B16 parent cells, as well as has highly aggressive ability, which will metastasize from the primary subcutaneous site to the lung in animal model. 46 The genetics and histological characteristics of murine melanoma are known to be similar to those of human melanoma. 47 PA exhibited growth inhibitory activity not only against mouse melanoma B16F10 cells, but also against human prostate cancer cells (PC3 and DU145), lung cancer cells (A549) and colorectal cancer cells (HCT116 and SW480), [25][26][27] suggesting that it had the potential of anti-cancer in both mouse and human cancers. The findings indicate that PA may be a potential compound for the development of novel drugs or adjuvants that can be used in combination with clinical drugs to improve side effects and drug resistance. More studies are needed, preferably using primary human melanoma, are needed to determine the potential and clinical application of PA in the therapy of metastatic melanoma.
In conclusion, this study provides evidence that PA exhibits potent inhibitory activity against melanoma cell proliferation in vitro and in vivo. PA treatment effectively induced cell cycle arrest at the G 0 /G 1 phase via downregulation of cyclin D1/CDK4 and triggered apoptosis via the activation of extrinsic and intrinsic pathways. It reduces the migratory ability of melanoma cells through the regulation of E-cadherin/vimentin and MMP-2/MMP-9 expression. In addition, the combination of PA and cisplatin showed synergistic inhibitory effects on colony formation and migration, and attenuated the development of drug resistance. These findings suggest that PA may be a potential anticancer candidate for the treatment of melanoma.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data presented in this study are available from the corresponding author upon reasonable request.