Quercetin can be a more reliable treatment for metastatic prostate cancer than the localized disease: An in vitro study

Abstract Quercetin is a plant flavonoid that has been recognized to have anti‐inflammatory, antioxidant and anti‐proliferative activities. This study aims to evaluate the inhibitory effects of quercetin against prostate malignancy in vitro and the underlying resistance mechanism. IC50 values of quercetin were determined by MTT assay. Annexin‐V/PI staining was used to measure the rate of apoptosis. DNA cell cycle was analysed by PI staining method. Real‐time PCR was performed to assess mRNA levels of OPN isoforms, VEGF isoforms, P53 and KLK2. Migration potential, proliferative capability and nucleus morphology of cells were evaluated by the scratch‐wound assay, colony‐forming assay and Hoechst staining, respectively. Quercetin significantly increased the apoptosis rate of PC‐3 and LNCaP cell lines, arrested the cell cycle at the sub‐G1/G1 phase, and reduced the migration potential and colony‐forming capability. Moreover, upregulation of apoptosis‐related genes and downregulation of genes involved in proliferation and angiogenesis was also observed. Although our results elucidated that quercetin has antitumor effects on PC‐3 and LNCaP, for the first time, we showed that quercetin treatment causes alterations in the expression of OPN and VEGF isoforms, which are cancer‐promoting modulators through various processes such as angiogenesis and drug‐resistance. Prostate malignant cells can dodge the anti‐carcinogenic properties of quercetin via modulation of OPN and VEGF isoforms in vitro. Therefore, quercetin acts as a double‐edged sword in prostate cancer treatment.

tumour cells become resistant to these treatments. Therefore, the disease relapses with a more aggressive phenotype, eventually leading to death. 3 Considering these challenges, optimizing classic treatment approaches and establishing new strategies are highly demanding.
Plants' secondary metabolites are considered great candidates to improve standard cancer treatments. Quercetin (3, 3′, 4′, 5-7 pentahydroxyflavone) is a bioactive phenolic compound that is abundantly found in fruits and vegetables such as apples, onions and tea. 4 Previous studies have shown that this compound has antioxidant, 5 anti-inflammatory 6 and anti-cancer properties. 7 This antitumor activity can be exerted via several mechanisms such as alteration of tumour metabolism, 8 inhibition of angiogenesis, 9 improvement of tumours' chemosensitivity, 10 inhibition of the epithelial-tomesenchymal transition, 11 decreasing tumour invasion potential through downregulation of matrix metalloproteinases, 12 induction of apoptosis and inhibition of anti-apoptotic pathways. 13,14 Despite its efficacy and minimal side effects, the clinical application of quercetin is limited due to its low bioavailability. 15 Many successful pieces of research have been performed, focusing on formulating quercetin using innovative nano-carriers to increase the bioavailability and therapeutic efficacy. 16,17 However, it is necessary to shed light on underlying mechanisms used by tumours to evade quercetinmediated anticancer effects to overcome post-treatment resistance and progression.

Osteopontin (OPN) is a secretory glycoprotein from the Small
Integrin-Binding Ligand N-linked Glycoprotein (SIBLING) family mainly produced by osteoblasts and osteoclasts to mediate the process of biomineralization. 18 It has been recognized that OPN also has oncogenic roles either as a prognostic biomarker or a significant regulator of tumour progression. [19][20][21][22] OPN has three splicing isoforms: OPN-a (full-length protein with seven exons), OPN-b and OPN-c (lack Exons 5 and 4, respectively). 23 Former studies have reported the overexpression of OPN-b and OPN-c in various cancer cell lines, mediating tumour survival, resistance to chemotherapeutic agents and, promoting angiogenesis. [24][25][26][27] However, the relationship between OPN and VEGF isoforms and quercetin in prostate cancer remains unclear. Therefore, our research aims to evaluate the antitumor potential of quercetin on LNCaP (Androgen-sensitive, metastatic to lymph node) and PC-3 cells (Androgen-insensitive, metastatic to bone), which represents different stages of prostate cancer. LNCaP represents localized disease, while PC-3 represents advanced disease. Quercetin-mediated modulatory effects on OPN and VEGF will be evaluated as a potential mechanism for tumour resistance and progression. supplemented with 10% fetal bovine serum (Gibco), 1000 units/mL Penicillin and 100 μg/mL streptomycin (Gibco) in a 5% CO 2 humidified incubator at 37°C.

| 3D cell colony formation assay
Cell lines in the treatment group and control group were seeded at the rate of 1.5 × 10 3 cells per well on six-well plates and incubated for 2 weeks. After 14 days, the bottom of the plate was coated by 2% agarose gel. Then, cells were mixed with the culture medium containing 0.7% agarose gel and poured on the gelcoated surface of the plate. The culture medium was changed every 4 days. After this time, cells were fixed with cold formaldehyde, washed with PBS, and stained with 0.1% crystal violet.
Calculation of the colony formation rate was done using ImageJ software.

| Flow cytometric measurement of apoptosis
Cell viability, apoptosis and necrosis were determined using an Annexin-V and PI (propidium iodide) kit according to the manufacturer's instructions. 28 After overnight incubation of cell lines in DMEM/10% FBS at 37°C, cells were exposed to various concentrations of quercetin for 2 days. Incubation in darkness for 15 min at 37°C was done after the addition of PI and Annexin-V, and then cells were analysed by flow cytometry device.

| Staining of treated and control cells with Hoechst dye (33342)
Apoptosis was further assessed by the Hoechst staining method.
Both cell lines were implanted in 24-well plates (3 × 10 5 cells per well), and incubated with various concentrations of quercetin for 48 h. Cells were treated with cold methanol (50 μL) for 20 min. After centrifugation, pellets were incubated with 100 μL PBS and 4 μL Hoechst dye for 20 min at 25°C in darkness, then observed under a fluorescence microscope (100× magnification).

| Migration potential analysis by Scratchwound assay
A vertical scratch was applied via pipette tip to the confluent PC-3 and LNCaP cells (about 85% confluency) and the plates were washed with serum-free medium twice. After overnight serum starvation, control and experimental groups were exposed to PBS and quercetin, respectively. Finally, cell imaging was performed at 24 h intervals. The cell migration rate was estimated by measurement of area among the two scratch edges in comparison to the control group.

| DNA cell cycle analysis
Quercetin-treated (48 h) and untreated cells were fixed using 70% cold ethanol for 24 h. After double wash with PBS, cells were incubated with RNase I and 500 μL PI for 30 min at 37°C. Cell detection was performed by flow cytometer. Flowjo software was used to analyse the data. Cell arrest at sub-G0/G1 was considered apoptosis.

| Gene expression analysis by real-time PCR
Total RNA extraction was performed using TriPure Isolation Reagent.
Colibri Microvolume Spectrometer was used to determine the RNA concentration. cDNA was generated using Takara cDNA synthesis kit. Real-time PCR was performed using QIAGEN's thermocycler with a total sample volume of 20 μL. The PCR reaction specificity confirmation was applied through melting curve analysis. GAPDH mRNA levels were considered as an internal control to estimate the relative expression levels by the 2 −ΔΔCT method. 29 Table 1 represents the nucleotide sequences of primers.

| Statistical analysis
All experiments were performed in triplicate and the data were presented as means ± SD. Statistical analysis was performed by anova and Student's t-test. Statistical significance was considered as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

| DISCUSS ION
Despite recent advances in the diagnosis and treatment, prostate cancer has remained a significant challenge in terms of prevalence and mortality. 1 Various treatments for prostate cancer still suffer from two major problems: side effects and tumour resistance. Naturallyoccurring compounds are perfect candidates to overcome these problems and improve treatment outcomes. 30  quercetin. Subsequently, the invasion potential of prostate cancer was also lessened. 12 Sun et al., also demonstrated that the combined use of metformin and quercetin synergistically suppressed the prostate cancer cells through the VEGF/PI3K/Akt axis. 35 According to the body of evidence from previous studies, human kallikrein-related peptidase 2 (KLK2), a protease that is exclusively expressed in the prostate gland, can facilitate prostate cancer progression and act as a biomarker. 36,37 It has been reported that during prostate cancer, upregulation of the KLK2 gene is associated with cell proliferation, migration, invasion, angiogenesis and apoptosis resistance of malignant cells. 38,39 Therefore, KLK2 has been used in many studies as a biological prostate cancer prognosis marker as well as a therapeutic target. Quercetin exposure resulted in a  24 It has been shown that OPN expression has a strong association with the expression of hypoxia-inducible factor 1 alpha (HIF-1a) and VEGF via Akt/ILK/NF-kB/ ATF-4/PI3K/αvβ3 integrin/ERK1/2 pathway. 40,41 According to previous studies, the role of VEGF isoforms in tumour microenvironments might be beyond angiogenesis and lymphangiogenesis. 29  There are detailed subjects that we aim to consider in our forthcoming research. For example, we would investigate the effects of

Control
Quercetin-treated Group

| CON CLUS ION
In the current study, we reconfirmed that quercetin has anti-

ACK N O WLE D G E M ENTS
Special thanks to Sina Hospital, Tehran University of Medical Sciences, Tehran, Iran.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Information, data, and photos will be provided if requested.