Vulpinic acid as a natural compound inhibits the proliferation of metastatic prostate cancer cells by inducing apoptosis

Lichen secondary metabolites have drawn considerable attention in recent years due to the limitations of current treatment options. Vulpinic acid (VA) obtained from Letharia vulpina lichen species exerts a remarkable cytotoxic effect on different cancer types. However, the therapeutic efficacy of VA in metastatic prostate cancer (mPC) cells has not been investigated. In the present study, we aimed to identify VA-mediated cytotoxicity in PC-3 mPC cells compared with control cells. After identifying the cytotoxic concentrations of VA, VA induced apoptosis was analyzed by Annexin V, cell cycle, acridine orange and propidium iodide staining and RT-PCR analysis. Our findings showed that VA significantly decreased the viability of PC-3 cells (p < 0.01) and caused a considerable early apoptotic effects through G0/G1 arrest, nuclear blebbing and the activation of particularly initiator caspases. Therefore, VA may be a potential treatment option for mPC patients. However, the underlying molecular mechanisms of VA-induced apoptosis with advanced analysis should be further investigated.


Introduction
Cancer is identified as an essential cause of death worldwide [1]. The cancer incidence is expected to rise to over 20 million new cases by 2025 [2]. Prostate cancer is the most diagnosed in men in Europe and is the third cancer type among cancer deaths [2]. In the early stages, hormone sensitive prostate cancer patients can be treated with surgery, radiotherapy and hormonal therapy. However, castration resistance is observed in nearly 10-20% of prostate cancer patients within 5 years and 84% of patients develop metastasis at the time of castration resistance [3,4]. Docetaxel is used as the treatment option for metastatic castration resistant prostate cancer patients [5]. However, drugresistance develops over time against chemotherapeutics used in prostate cancer patients. Therefore, future studies should focus on researching new, less toxic and more potent drug candidate molecules that affect cancer cells.
In recent studies, the effectiveness of natural products on cancer cells has been determined due to their abundant and excessive extraction from nature and non-toxic effects [6,7]. Within the scope of drug development studies, 175 anti-cancer drugs were developed between 1940 and 2010. Approximately 50% of the anti-cancer drugs developed are of natural origin or derived from natural products [8,9]. Natural products can be obtained from plants, animals, marine organisms, lichens, fungi and microorganisms [10]. Plants synthesize many bioactive secondary metabolites such as alkaloids, flavonoids, phenolics, terpenoids, steroids, tannins, and essential oils [11]. Lichens are a powerful biological organism for the synthesis of many economically, pharmaceutically and strategically important bioactive secondary metabolites.
Vulpinic acid (VA) is one of the secondary metabolites of lichen. The chemical formula of VA is C 19 H 14 O 5 and VA has antimicrobial, antifungal and anti-cancer activities. The anti-proliferative effect of VA isolated from Letharia vulpina lichen species on HepG2, NS2OY and HUVEC cells has been determined [12]. In our previous study, the anti-proliferative and apoptotic effects of VA on different breast cancer cells (MCF-7, MDA-MB-231, BT-474,  SK-BR-3) and epithelial cells (MCF-12A) are revealed [13]. With a similar approach, the anti-cancer activity of VA on different cancer cell lines (CaCo2, HepG2, Hep2C, RD, Wehi) has also been determined in another previous study [14]. The results obtained show that VA could be evaluated as a cancer-focused promising drug candidate molecule.
In this context, we aimed to develope VA-mediated new approaches for the treatment and prevention of metastatic prostate cancer, one of the aggressive cancer types, and to elucidate the apoptosis mechanism after treatment with VA application. Herein, we showed that VA selectively killed prostate cancer cells without significant side effects on noncancerous epithelial cells and we also provided evidence suggesting the VA induced apoptosis through extrinsic pathways via induction of caspases and Bax mRNA level.

Cell culture
The human metastatic prostate cancer cells (PC-3) and human umbilical vein endothelial cells (HUVEC) were obtained from the American Type Culture Collection (ATCC, Rockville, USA). The PC-3 cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640, Gibco, USA) cell culture medium and HUVEC cells were seeded in Dulbecco's modified eagle medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (Gibco, Invitrogen, USA). The cells were incubated in a humidified incubator at 37 °C with 5% CO 2 . xCELLigence assay PC-3 and HUVEC cells (1 × 10 4 cells/well) were cultured in e-plates (ACEA Biosciences Inc., San Diego, CA, USA) and placed into the RTCA xCELLigence instrument (Roche Diagnostics, Mannheim, Germany). The cells were exposed to different concentrations of VA (1.56, 3.12, 6.25, 12.5, 25, 50 and 100 µM) and cellular impedance was measured in intervals of 15 min for 96 h. The commonly known anticancer drug Cabazitaxel (1, 5 and 10 nM) was used to compare the anti-proliferative effect of VA. The time-dependent cell index (CI) graph was analyzed by xCELLigence RTCA S16 Instrument (xCELLigence RTCA, Roche, Germany). According to the obtained results, the value of IC 25 , IC 50 and IC 75 in the most effective exposure time was calculated and used for further analysis.

Annexin V analysis
The cells (1 × 10 5 cells/well) were treated with IC 25 , IC 50 and IC 75 dose of VA for 48 h in 12-well plates. Afterward, the cells were harvested and resuspended with the Muse™ Annexin V and Dead Cell reagent. The stained cells were analyzed using the Muse™ Cell Analyzer (Millipore, Germany) and the cells were classified into four groups: live, early apoptotic, late apoptotic and necrosis.

Cell cycle
The cells (5 × 10 5 cells/well) were exposed to IC 25 , IC 50 and IC 75 dose of VA for 48 h. Following incubation, the cells were fixed in 70% ethyl alcohol (EtOH) for 3 h. Afterward, Muse™ Cell Cycle Kit (Merck Millipore, Germany) was added into each tube and incubated for 30 min in the dark and analyzed by the Muse™ Cell Analyzer.

Acridine orange (AO) and propidium iodide (PI) staining
The cells (2 × 10 5 cells/well) were treated with IC 25 , IC 50 and IC 75 dose of VA for 48 h. After incubation, the cells were fixed with 4% paraformaldehyde for 30 min and AO/ PI staining was added into each well and incubated for 30 min in dark conditions. The cells were examined with EVOS FL Cell Imaging System (Thermo Fisher Scientific, USA).

Multi-caspase analysis
The cells were cultured in 6-well plates and then treated with IC 25 , IC 50 and IC 75 dose of VA for 48 h. After incubation, multi caspase assay (caspase-1, 3, 4, 5, 6, 7, 8, and 9) was performed according to kit instruction. Finally, the cells were analyzed by the Muse™ Cell Analyzer.

Real-time (RT) PCR analysis
PC-3 cells (5 × 10 5 cells/well) were cultured in 6-well plates and then treated with IC 50 dose of VA for 48 h. RNA quantity/quality was measured with a NanoDrop 1 3 1000 ND-Spectrophotometer (Thermo Fisher Scientific, Waltham, USA). One microgram of the total RNA from each sample was converted to cDNA using Transcriptor High Fidelity cDNA Synthesis Kit (miScript® II RT Kit, Qiagen). The level of gene expression was determined by Human Apoptosis Primer Library (Real Time Primers, US) by using LightCycler 480 PCR (Roche, Germany). GAPDH is used as a housekeeping gene which is used for normalization. All qRT-PCR reactions were performed in three independent biological triplicates.

Statistical analysis
The target gene transcripts was normalized to GAPDH and set relative to control samples according to the 2 -ΔΔCT method [15]. The result of gene expression fold change was applied one-way ANOVA. The Fisher's significance level (p ˂ 0.05) was performed. Graphpad Prism 8.0 software was used to represent data on the graph.

VA decreased the viability of PC-3 cells
The effect of VA on the viability of the cells was tested by the RTCA xCELLigence system for the first time in this study. RTCA xCELLigence system was enabled to measure with electronic impedance for determining detailed monitoring the cells continuously and quantitatively. The IC 50 value of VA is calculated based on CI at every measuring point of study. The obtained IC 50 value of VA by using the RTCA xCELLigence system was 27 µM for 48 h obtained from the sigmoidal dose-response formula in software (Fig. 1A). Cabazitaxel was 10.9 µM for 48 h on PC-3 cell (Fig. 1B). Additionally, HUVEC cells were treated with all examined concentrations of VA. All concentrations of VA increased compared with control in HUVEC non-cancerous cell by using the RTCA system (Fig. 1C). It was showed that VA inhibited the proliferation of PC-3 prostate cancer cells. However, the lower concentrations of VA did not exert a cytotoxic effect on non-cancerous epithelial cell lines (HUVEC). Therefore, VA could be a better therapeutic option than cabazitaxel in terms of dose concentration.

Evaluation of VA induced apoptotic cell death in metastatic prostate cancer
The induction of VA-mediated apoptosis in castration resistant metastatic prostate cancer cells was assessed by Annexin V and cell cycle analysis (Figs. 2 and 3). Our findings demonstrated that VA caused a considerable increase in the early apoptotic cell death in PC-3 cells dose-dependently (Fig. 2, p < 0.01). Following treatment with the IC 25 , IC 50 and IC 75 dose of VA, the total apoptotic death rate increased from 2.6 to 31.9%, 48.9% and 60.7%, respectively in PC-3 cells. Furthermore, the total of apoptotic cell death was detected as 15.1%, 25.1% and 34.7% at the concentration of IC 25 , IC 50 and IC 75 , respectively in HUVEC cells in Fig. 2.
VA-mediated cell cycle arrest was also analyzed in our study. As shown in Fig. 3, VA treatment induced G0/G1 arrest in both PC-3 and HUVEC cells. In PC-3 cells, the accumulation of cells in G0/G1 arrest significantly increased to 62.8%, 68.8% and 74.3%, at the concentration of IC 25 , IC 50 and IC 75 , respectively (p < 0.01). On the other hand, the population of cells in G0/G1 phase was remarkably increased to 64.6%, 70.6% and 79.4%, respectively in VA treated HUVEC cells compared to control cells (51.5%). Therefore, VA treatment could result in significant apoptotic cell death by inducing G0/G1 phase arrest in metastatic prostate cancer cells.

Observation of VA induced apoptotic cell death by AO/PI staining
AO/PI staining results revealed that VA treatment changed particularly nuclear morphology in metastatic prostate cancer cells (Fig. 4). In PC-3 cells, condensation of DNA and markedly the formation of nuclear bleb through degradation of nuclear lamina were observed dose-dependently compared with control cells. However, similar nuclear damages were also observed in HUVEC cells after exposure to the increased concentration of the VA.

VA induced the activation of multiple-caspases and changed the expression of target genes
We performed multi-caspase analysis to confirm further VA induced apoptosis (Fig. 5A and B). At IC 25 , IC 50 and IC 75 concentration, VA treatment resulted in a significant increase in multi-caspase activity with especially caspase positive/ dead cells percentage (8.1%, 12.7% and 29.9%, respectively) (p < 0.05). Furthermore, we used real-time PCR using Human Apoptosis Primer Library to determine some apoptotic gene expression patterns after applying VA in PC-3 cell. In RT-PCR analysis, the IC 50 value of VA was selected as an optimum concentration. The target gene expression was demonstrated in Fig. 5C. In the PC-3 cell, VA significantly increased CASP1 (p < 0.05), CASP4 (p < 0.001), CASP5 (p < 0.01), CASP2, CASP8 (p < 0.05), CASP8AP2 (p < 0.05), CASP10 (p < 0.05), CASP3 (p < 0.01), CASP7 (p < 0.05) and Bax (p < 0.001) gene expression compared with the control. Bcl-2 gene expression was significantly down-regulated after treatment with VA in PC-3 cells compared with control. Our findings were consistent with the results of multi-caspase activity.

Discussion
Herein, we investigated the potential therapeutic effects of VA on castration resistant metastatic prostate cancer cells. Our findings showed that VA treatment could result in apoptotic cell death through G0/G1 arrest, the activation of caspases, nuclear blebbing and particularly the activation of apoptotic cell death initiators (caspase 1, caspase 2 and Secondary metabolites of lichens have great attention in preclinical cancer studies due to their anti-inflammatory, anti-proliferative, and anti-neoplastic effects [12,16,17]. After isolation of several seconder metabolites such as usnic acid, atronarin, gyrophoric acid, barbaric acid, hypostictic acid and vulpinic acid (VA) obtained from different lichens and/or extraction of lichens, their potential anti-cancer effects have been determined in various types of cancer cell lines [12,17,18]. Among them, there is a limited number of studies investigating the potential therapeutic efficacy of VA in cancer cells [13,14,19,20]. The study of Kılıc et al. (2018) states that VA exhibits significant cytotoxicity in breast cancer cell lines (MCF-7, MDA-MB-231, BT-474, SK-BR-3) compared to MCF-12A control cells and triggers apoptosis in the cells [13]. Furthermore, the expression level of p53 increased to 14-fold in SK-BR-3 cells compared to control cells following treatment with IC 50 concentration of VA [13]. In another study, the cytotoxic effects of VA on CaCo2, HepG2, Hep2C, RD, Wehi and normal cells (Vero and L929) have been assessed at a molecular level. Their findings show that IC 50 dose of VA is different in terms of the features of cells and the expression level of Bax is significantly increased in all cell lines due to its apoptotic effects [14]. The study of Kim et al. (2017) states that the isolated VA from Pulveroboletus ravenelii as an edible mushroom mediates the cytotoxic effects of P. ravenelli on different human cancer cells, including lung adenocarcinoma, pancreatic ductal adenocarcinoma and hepatocellular carcinoma cell lines through the induction of apoptosis [20]. Furthermore, the anti-angiogenic and antiproliferative effects of VA on HEPG2, NS2oY and HUVEC cells compared with usnic acid (UA) have been determined. According to their findings, both UA and VA exhibit more cytotoxic activity in cancer cell lines than normal cell lines. However, VA exerts more anti-angiogenic activity than UA [20]. In the present study, we investigated VA-mediated cytotoxicity in castration resistant metastatic prostate cancer cells due to highly aggressive behavior including metastatic and angiogenic potential of prostate cancer cells, for the first time. Our findings demonstrated that VA significantly decreased the viability of PC-3 cells (p < 0.01) and induced early apoptosis in dose-dependently through G0/G1 arrest and the formation of nuclear bleb with less toxicity in HUVEC normal cells. Moreover, considerably higher expression levels of pro-apoptotic markers were analyzed in PC-3 cells after treatment with IC 50 value of VA (p < 0.05). Interestingly, a nearly 25-fold increase in the expression of Caspase-1 level was determined in PC-3 cells treated with VA compared to other apoptotic markers. The caspase 3 and 9 protein levels are low in PC-3, DU-145, TSU-Pr1m and LNCaP cells, while lower expression of caspase-1 is observed in DU-145 and LNCaP cells [21]. Therefore, the loss of caspase-1 is an essential step in prostate tumorigenesis. Additionally, ◂ the over-expression of Caspase-1 induces TGF-β and Fasmediated apoptosis and enhances radiosensitivity in prostate cancer cells [22]. On the other hand, caspase-1 proteolytic activity is mediated by inflammasome and some interleukins (IL-1β and IL-18), which are substrates of caspase 1 [23]. Therefore, a higher activity of caspase-1 can be associated with the apoptotic and anti-inflammatory effects of VA on prostate cancer cells in our study. However, further investigations should be performed to elucidate the underlying mechanisms of the VA induced caspase-1 activity in the tumor microenvironment. Additionally, the expression levels of Caspase 2, 3, 5, 8, and Bax were higher in VA treated cells compared to control cells due to its apoptotic effects via the down-regulation of Bcl-2 level and the activation of multiple caspases. In the study of Taghiyev et al. (2011), the activation of Caspase-2 mediates AR activity in LNCaP prostate cancer cells [24]. Briefly, our findings showed that VA regulated the activation of initiators caspase expression levels and extrinsic apoptotic mechanism. However, the underlying molecular mechanism of VA induced apoptosis and its associated signaling pathways as well as the function of caspase activation, need further investigations at particularly protein level.

Conclusion
In our study, VA exhibited significant anti-proliferative and apoptotic effects on castration resistant metastatic prostate cancer cells by activating initiator caspases and G0/G1 arrest. Therefore, VA could be a new therapeutic target as a seconder metabolite for effective treatment in advanced prostate cancer. However, further preclinical studies are needed to investigate the efficacy of VA in different types of prostate cancer cells at molecular level and to determine the potential combined effects of VA with different chemotherapy drugs.