Cell mitosis and proliferation play an important role in the progression of tumors. Hence, cell cycle arrest and proliferation inhibition are effective measures for cancer treatment. Quercetin can play this role in prostate cancer. We explored the effect of quercetin on the proliferation of human prostate cancer PC-3 and LNCaP cells treated with varying doses and found that the inhibition rate demonstrated a dose-dependent increase. IC₅₀ values of quercetin were found to be 22.12 μM for PC-3 and 23.29 μM for LNCaP cells. Quercetin treatment not only resulted in an increase in the G2/M phase population in both PC-3 and LNCaP cells, but also increased the S phase population in PC-3 cells (4).

Liu et al treated human prostate cancer PC-3 cells with quercetin at various doses (50–200 μM) for 24 and 48 h and found that cell viability was significantly decreased in a time- and dose-dependent manner. It was attributed to induction of G0/G1 (31.4–49.7%) and sub-G1 (19.77%) cell cycle arrest which was caused by downregulation of cyclin D and E, CDK2, cdc25c and upregulation of p21, p53, p18 and p27 (21). In PPC1 prostate carcinoma cells, quercetin at a high dose arrested the cell cycle and inhibited proliferation. However, the p53 status should be taken into consideration (22). Quercetin also displayed proliferation inhibition in a dose-dependent manner in PC-3 cells at a non-cytotoxic concentration, during which endoplasmic reticulum (ER)-mediated and ER-independent pathways as well as cell cycle inhibition induced by cyclin D1 and E downregulation may be the vital factors (23). Other studies obtained the same results for quercetin and suggested that anti-proliferation was achieved through modulation of NO production (24–27).

Apoptosis is defined as programmed cell death and plays an important role in maintaining stabilization of cell homeostasis. It is divided into death receptor (DR)-mediated extrinsic and mitochondrial-mediated intrinsic pathways, and they both activate the common ̔executor’ caspase-3 leading to cell apoptosis. As insufficient apoptosis is a critical cause of tumorigenesis, many drugs treat cancers by inducing apoptosis (28). We conducted research to investigate the effect of quercetin on PC-3 and LNCaP cells and found that quercetin induced apoptosis by increasing pro-apoptotic Bax and by decreasing anti-apoptotic Bcl-2 protein resulting in a significant decrease in the Bcl-2/Bax ratio (4).

After PC-3 cells were treated with quercetin, in addition to a decrease in anti-apoptotic Bcl-2 and an increase in pro-apoptotic Bax, ER stress-associated proteins such as GRP78, ATF-4α and IRE-1α were also increased, followed by direct activation of the caspase cascade leading to subsequent apoptosis through the mitochondrial pathway and ER stress (21). Quercetin was found to enhance extrinsic apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in DU-145 cells either through DR5 upregulation (29) or survivin downregulation via deacetylation of histone H-3 mediated by ERK in PC-3 and DU-145 cells (30) or dephosphorylation of AKT in LNCaP and DU-145 cells (31).

From the previously published studies, it can be concluded that quercetin induces apoptosis of prostate cancer mainly by regulating Bax, Bcl-2 and the Bcl-2/Bax ratio, namely through mitochondrial-mediated intrinsic pathway, and it can also mediate the extrinsic pathway (23,32,33). Induction of apoptosis in various types of prostate cancer cells by quercetin which may be the main property of its anti-prostate cancer effects has been widely studied and is gaining more and more recognition (34–37).

AR, a nuclear receptor belonging to a superfamily of ligand responsive transcription, regulates physiological actions of androgen (38) and is nearly expressed in all types of prostate cancer (39). Since the relationship of AR and the development and progression of prostate cancer has been verified, targeted therapy of AR is now considered as a promising measure for controlling prostate cancer progression (40–42). We treated LNCaP cells with quercetin and found that AR protein was reduced in a dose-dependent manner after a designated time. Moreover, the regulated tumor markers, PSA and hK2, were inhibited, and regulated genes such as PSA, NKX3.1 and ornithine decarboxylase (ODC) mRNA were downregulated. These findings indicate that quercetin not only decreases AR expression but also impairs the function of AR and has the potential to serve as a chemotherapeutic drug for prostate cancer (3).

Quercetin treatment reduced expression of AR and then increased caspase-3/-7 causing subsequent anti-proliferation and apoptosis in LNCaP cells (43). It also reduced expression and impaired the function of AR through c-Jun or Sp1 which either interacted directly with AR or formed c-Jun/Sp1/AR protein complex (42,44–46). Other anti-prostate cancer functions of quercetin through AR include retardation of DNA synthesis and modulation of the AR signaling pathway. The common findings included decreased expression and impaired function of AR and finally, the inhibition of prostate cancer growth (47–49).

The PI3K/AKT signaling pathway is upregulated in 30–50% of prostate cancer cases. When phosphorylated at serine 473, AKT is activated into phosphorylated AKT (pAKT) which plays a vital role in the proliferation, survival and progression of prostate cancer and helps inhibit apoptosis by phosphorylating downstream substrates (50). Recent research reported that in the progression from androgen-dependent to androgen-independent status, the PI3K/AKT signaling pathway may be of great importance. Even though androgen is at a very low level, it can maintain the high proliferation of prostate cancer cells (51). Therefore, it is speculated that PI3K/AKT inhibitors can effectively treat prostate cancer.

Recently, a latent PI3K inhibitor prodrug was generated using a quercetin analog and a peptide that could cleave PSA. When the inhibitor was activated, the PI3K/AKT signaling pathway was inhibited and apoptosis was induced resulting in the cell death of androgen-independent prostate cancer C4-2 cells that could secret PSA (52). In PC-3 cells, quercetin significantly reduced pAKT levels and reversed its anti-apoptotic effect preventing tumor cells from infinite proliferation. The results were the same for LNCaP and DU-145 cells (23,31–33). In addition, the AKT signaling pathway was found to contribute to angiogenesis. Yet, when it was suppressed by quercetin, angiogenesis was effectively suppressed (53).

Angiogenesis is the process of new blood vessel formation from the pre-existing vascular system and is regulated by two angiogenic factors: vascular endothelial growth factor (VEGF) and hypoxia inducible factor (HIF) α (54). Blood vessels are abundant in tumor tissues as tumors themselves can induce neovascularization. This provides tumors with enough oxygen and nutrients and ensures their development and progression (55–57). Anticancer drugs inhibit angiogenesis by targeting angiogenic factors (58,59).

We administered varying doses (0–100 μM) of quercetin to a rhesus choroid-retina endothelial cell line (RF/6A) and found that endothelial cell proliferation, migration and tube formation were significantly inhibited after incubation for 24, 48 and 72 h. Our experimental results concluded that quercetin inhibited angiogenesis in vitro (60). Pratheeshkumar et al carried out in vitro and ex vivo experiments to examine the effect of quercetin on angiogenesis and showed that quercetin not only greatly inhibited angiogenesis in vitro by influencing the critical processes of proliferation, migration, invasion and tube formation of endothelial cells, but also inhibited angiogenesis ex vivo via reducing vascularized structure and microvessel outgrowth. When PC-3 cells were treated with quercetin, VEGF secretion and cell viability were markedly decreased in a dose-dependent manner, and this occurred by having a negative impact on the AKT/mTOR/P70S6K pathway (53). As for LNCaP cells, quercetin inhibited angiogenesis by decreasing HIF-1α accumulation and VEGF secretion (61).

ERK-1/2, JNK and p38-MAPK are the three most important components of the MAPK signaling pathway (62). In prostate cancer, the ERK pathway is activated and is associated with advanced stages and high invasive property (63,64). JNK and p38 play an important role in promoting apoptosis and negatively regulating tumor cell growth (65). Quercetin treatment in PC-3 cells decreased the ERK-1/2 level and increased JNK expression by which cancer apoptosis was prompted and tumor growth was inhibited (23). As another important component of the MAPK signaling pathway, p38-MAPK was increased by quercetin in PC-3 cells contributing to the inhibition of cancer survival and proliferation (66).

The IGF/IGF-R axis is regarded as a critical element in the initiation and development of prostate cancer, and overexpression of insulin receptor in human prostate cancer has been identified (67). Androgen-independence and progression of prostate cancer are related with IGF/IGF-R overexpression (68). Quercetin decreased IGF-I, IGF-II, IGF-IR mRNA and IGF-IRβ protein in PC-3 cells and could be used for androgen-independent prostate cancer treatment (33). Another two studies showed that quercetin increased insulin-like growth factor-binding protein-3 (IGFBP-3) which had high binding affinity with IGFs causing marked reduction. In this manner, the Bax/Bcl-2 protein ratio was modulated and apoptosis was induced via a p53-independent manner (69,70). Moreover, in the AT6.3 rat prostate cancer cell line, quercetin acted via a reduction in insulin-like growth factor-1 (71).

EGF can promote EMT in tumors making them acquire high invasiveness and this also occurs in prostate cancer suggesting the pivotal role of EGF in mediating progression and metastasis of prostate cancer. Bhat et al showed that quercetin reversed EMT in PC-3 cells induced by EGF which prevented tumor cells from invading and migrating. This was achieved through a decrease in EGF-induced transcriptional repressors such as Snail, Slug and Twist and inhibition of the EGFR/PI3K/AKT/ERK1/2 pathway (72). Tang et al also demonstrated that quercetin inhibited EMT and expression of related molecules (73). Therefore, targeting EGF by quercetin can effectively prevent prostate cancer from metastasizing.

Quercetin binds with the C-terminal region and interacts with hnRNPA1 leading to cytoplasmic retention manifested as abnormal shuttle between the nucleus and cytoplasm, which greatly impairs the function of hnRNPA1 and inhibits PC-3 cell growth (74).

Apart from the possible mechanisms elaborated above involved in quercetin treatment of different prostate cancer cell lines, there also exist other mechanisms reported by researchers.

Quercetin inhibited nuclear factor (NF)-κΒ-mediated transcriptional activity of COX-2 promoter (75) and acted on urokinase-type plasminogen activator (uPA) and its receptor resulting in inhibition of factors related to survival and proliferation in PC-3 cells (66). Quercetin restrained prostate cancer stem cells isolated from PC-3 and LNCaP cells from invading and migrating (73). Quercetin also used Hedgehog signaling pathway as a direct or indirect target to antagonize TRAMP-C2 cells (76), increased tumor suppressor p53 (48), downregulated oncogenes and cell cycle genes (77), and stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion causing immune therapy in PC-3 cells (78).

What is more, quercetin also reduced heat shock protein (Hsp) 90, 70 and 72 expression (79–82), downregulated matrix metalloproteinases 2 and 9 protein (83), inhibited CYP1 cytochrome P450 enzymes (84) and fatty acid synthase activity (85), and ErbB-2 and ErbB-3 expression (86) in prostate cancer cell lines.

Contrary to many in vitro studies, there are only a few in vivo studies of quercetin on prostate cancer. However, quercetin has begun to be used in vivo and these preclinical results will lay a good foundation for subsequent clinical trails.

Pratheeshkumar et al injected 5×10⁶ PC-3 prostate cancer cells into male 6-week-old BALB/c nude mice. When the tumor volume reached ~100 mm³, the mice in the treatment group received quercetin at 20 mg/kg/day intraperitoneally. Treatment with quercetin significantly inhibited tumor growth as compared to the vehicle control 15 days later. Western blot and immunohistochemical analyses exhibited that quercetin inhibited angiogenesis through the AKT/mTOR/P70S6K signaling pathway mediated by VEGFR-2 (53). Ma et al used CWR22 prostate cancer cell xenograft tumors to evaluate the therapeutic effect of quercetin and found that quercetin at 200 mg/kg reduced tumor volume by 51.1%, which was due to a decrease in VEGF121 and VEGF165 that negatively regulated vascular formation (87).

After intervention of a 0.4% quercetin diet for 6 weeks, the growth of androgen-sensitive LAPC-4 prostate cancer cell xenograft tumors in male SCID mice was inhibited by 15%. Subsequent biomarker analysis revealed that this was attributed to apoptosis induction represented as increased Bax and Bax/Bcl-2 ratio, proliferation inhibition manifested as decreased Ki67, and downregulation of pAKT, PSA and AR (88). Ma et al also demonstrated tumor inhibition by quercetin at the dose of 200 mg/kg which they attributed to proliferation suppression caused by modulation of the phosphorylation of cdc-2 and cyclin Bl (87). The same effectiveness and mechanism were observed in another study using 50, 100 or 150 mg of quercetin to reduce prostate weight in male Sprague-Dawley rats. Phospho-MEK1/2, phospho-MAPK, p15, p21 as well as p27 were also involved (89).

When quercetin at 150 mg/kg was administered intraperitoneally in prostate cancer PC-3 and DU-145 xenograft tumor models, it suppressed xenograft tumor growth attributed to the antagonization of HSP72 expression (82).

When quercetin was combined with heat treatment in prostate cancer PC-3, LNCaP and JCA-1 cell lines, due to the reduction in HSP70 or HSP72 protein expression and decreased tolerance of cells to heat treatment, it greatly enhanced the heat-induced inhibitory impact on proliferation and the stimulative effect on apoptosis in prostate cancer (81,82). Notably, low-frequency ultrasound sensitized prostate cancer cells to quercetin therapy and increased its inhibitory effect, although the reason remained unclear (96).