The anti-tumoral role of Hesperidin and Aprepitant on prostate cancer cells through redox modifications

Prostate cancer is the second prevalent cancer in men. While the anti-cancer effect of Hesperidin and (Aprepitant) AP on prostate cancer cells is well documented, their combined effect and their mechanism of action are not fully investigated. Therefore, this study aimed to investigate the anti-cancer effects of Hesperidin and AP alone and in combination on prostate cancer cells. PC3 and LNCaP cell lines were treated with Hesperidin and AP alone and in combination. The Resazurin test was used for assessing cell viability. The ROS (reactive oxygen Species) level, P53, P21, Bcl-2, and Survivin gene expression were assessed. Also, a trypan blue assay was done. Hesperidin and AP reduced cell viability and increased apoptosis in PC3 and LNCaP cells. The ROS level reduced after treating the PC3 and LNCaP cells with AP with or without Hesperidin. P53 and P21 gene expression increased after treatment with Hesperidin with or without AP compared to the untreated group in the PC3 cell line. Bcl-2 and Survivin gene expression decreased with AP with or without Hesperidin in the PC3 and LNCaP cells. The current study showed the synergic anti-cancer effect of Hesperidin and AP in both PC3 and LNCaP cell lines.


Introduction
Prostate cancer is the second most common frequent malignancy and the fifth most common cause of cancer-related death among men worldwide (Bray et al. 2018;Ferlay et al. 2018).The current treatment for prostate cancer is radical prostatectomy, with radio or chemotherapy followed by androgen deprivation therapy (Rydzewska et al. 2017).However, their adverse effects, cancer relapse, and tumor resistance have limited their efficacy (Nakazawa et al. 2017).Many investigations tried to identify the mechanism behind prostate cancer but there are still unknowns.Several recent therapies were introduced for prostate cancer including monoclonal antibodies, vaccines, and other types of targeted drugs (O'Neill et al. 2015).However, these studies are in the initial steps and the results need to be improved.Therefore, it's necessary to find a new effective therapy for prostate cancer with no adverse effects on healthy cells.
Aprepitant (AP) is already approved by the Food and Drug Administration (FDA) as an effective agent for the prevention of chemotherapy-induced nausea and vomiting (Muñoz and Coveñas 2020).Previous studies showed neurokinin-1 receptor (NK1R) and its agonist, substance P (SP), are overexpressed in different types of cancer cells including gastric, glioblastoma, larynx, colon, pancreatic, and prostate cancers (Ghahremanloo et al. 2021;Cussenot et al. 1996).Besides, SP/NK1R axis is shown to have an important role in the progression, angiogenesis, and metastasis of various cancer cells (Esteban et al. 2006).With regard to these features, pharmacologic NK1R inhibition has become an important strategy for new cancer treatments.AP is a highly specific NK1R antagonist and its anti-cancer effects against different cancer cells including prostate cancer are shown previously (Ebrahimi et al. 1869).Besides, no adverse effects on healthy cells were found even after the administration of a large dose of AP (Muñoz and Rosso 2010).

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In addition, phytochemical treatments have been used as an excellent cancer treatment due to easy extraction from plants, cost-effectiveness, and low side effects (Zhang et al. 2020).A flavanone largely extracted from citrus fruits called Hesperidin has been shown to have considerable beneficial effects on the human body (Tanwar and Modgil 2012).Previous in vivo and in vitro investigations presented the anti-inflammatory, antioxidant, and neuroprotective role of Hesperidin (Li and Schluesener 2017).The anti-cancer effect of Hesperidin has been shown in different cancer cells including both androgen-dependent and -independent prostate cancer cells (Lee et al. 2010;Ning et al. 2020).
While the anti-cancer effect of Hesperidin and AP on prostate cancer cells is well documented, their combined effect, as well as their mechanism of action are not fully understood.Therefore, the present study aimed to investigate the anti-cancer effects of Hesperidin and AP alone and in combination on prostate cancer cells.In addition, their effect on reactive oxygen species (ROS), tumor suppressors (P53 and P21), and antiapoptotic genes (Bcl-2 and Survivin) were evaluated.

Cell culture
The present study was performed at the Mashhad University of Medical Science, Mashhad, Iran Two different prostate cancer cell lines, including PC3 and LNCaP, and also Human fetal foreskin fibroblast cells (HFF-1) were purchased from the National Cell Bank of Institute Pasteur of Iran.PC3 and LNCaP cells were cultured in RPMI 1640 medium, and HFF-1cells were grown in Dulbecco's Modified Eagle Medium (DMEM).One percent antibiotics (penicillin/streptomycin) and 10% heat-inactivated fetal bovine serum (FBS) were added to the cell cultures.Cell lines were kept at 37 • C humidified incubator with 5% CO 2 .

Drugs
AP was purchased from Sigma-Aldrich Company (St.Louis, MO, USA) and dissolved in ethanol.Also, Hesperidin was bought from a local source (Golexir Pars) and dissolved in dimethyl sulfoxide (DMSO).

Resazurin cell viability assay
Resazurin cell viability tests were performed to investigate the cell toxicity of Hesperidin and AP.Resazurin cell viability assay is on the basis of the magnitude of resazurin (nonfuorescent) transformation to resorufn and dihydro-resorufn (highly fuorescent) by the metabolically active cells.There is a direct association between the rate of dye reduction and the number of viable cells in a sample (O'brien et al. 2000).Briefly, a 96-well plate was used to seed 2.5 × 10 4 cells in a volume of 100 µL and treated with different concentrations of AP including 0 (control), 5,10,20,50,70,and 90 μM,and Hesperidin 0 (control),10,50,100,200,300 and 500 μM for 24 and 48 h.Then, after removing the medium, each well received 10 µL resazurin solution (0.01 mg/mL dissolved in phosphatebufered saline; Sigma-Aldrich) for 3 h at 37 °C, under 5% CO2 and protected from light.By using a microplate fuorimeter, the colorimetric assays were done under the emission and excitation wavelengths of 570 and 600 nm, respectively.The results were presented as percentage survival rates by comparing the treated cell absorbance with the untreated control.the Graph-Pad Prism® 6 software was used for calculating IC50.

ROS level assay
Based on the manufacturer's protocol, the Intracellular ROS level was assessed by using a 2′,7′-Dichlorodihydrofluorescein diacetate (DCFDA) cellular ROS detection assay.According to the protocol, 20 μM DCFDA was added to cells and after 24 h of incubation, DCFDA was washed.Afterward, the rewashed cells were treated with AP at a concentration of 10 µM, Hesperidin at concentrations of 50 and 100 µM, and the combination of 10 µM AP + 50 µM Hesperidin and 10 µM AP + 100 µM Hesperidin for 24 h.Tert-butyl hydroperoxide (TBHP) was used as a positive control.Finally, the relative fluorescence intensity was measured with the fluorescence plate reader Perkin Elmer.

RNA extraction and quantitative real-time PCR (qRT-PCR)
The RNA extraction kit (Pars Tous biotechnology, Iran) was used for RNA extraction from cultured cells including PC3 and LNCaP.According to the manufacturer's instructions, complementary DNA (cDNA) Synthesis Kit (Pars Tous biotechnology, Iran) was used to reversely transcribe the RNA to cDNA.LightCycler ® 96 RT-PCR system (Roche, USA) was used for qRTPCR amplifications with a fluorogenic dye detection system (SYBR green).One of the most common housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was applied as an internal reference gene and ΔΔCT method was used for the analysis of the relative changes with Graph-Pad Prism software (version 6.0).qRTPCR was performed for Bcl-2, Survivin, P53, and P21 primers which were purchased from Pishgaman (Pishgaman Co., Tehran, Iran).

Trypan blue assay
PC3 and LNCaP cells were treated with the aforementioned doses of Hesperidin and AP for either 24 or 48 h.Next, the viable cell density was done with 0.4% trypan blue staining as described (Javid et al. 2020).Trypan blue was used for cell staining.Then, an optical microscope was used for counting the stained (dead) and unstained (viable) cells.According to the following formula, the viable cell density was determined: % cell viability = (viable cell count/total cell count) × 100.

Statistical analysis
The experimental data were presented as mean ± standard error of the mean.The ANOVA test followed by Bonferroni's t-test was applied for statistical analysis.All the data were analyzed triplicate compared to the untreated control group.The statistically significant level was considered p-value < 0.05. the GraphPad Prism® 6.0 software (San Diego, CA,USA) for Windows was used for statistical analysis.

The effect of AP and Hesperidin on cell viability of PC3 and LNCaP cell lines
The PC3, LNCaP, and HFF-1 cell lines were treated with increasing concentrations of AP (5, 10, 20, 50, 70, and 90 µM) and Hesperidin (10, 50, 100, 200, 300, and 500 µM) for 24 h.As shown in Figs. 1 (a and b), IC50s were 212 and 235 µM in PC3 and LNCaP cell lines after 24 h of Hesperidin inoculation, respectively.In addition, IC50s were 18.30 and 22.35 µM in PC3 and LNCaP cell lines after 24 h of AP, respectively (Figs. 2 (a and b)).Furthermore, as shown in Fig. 3, the IC50 of the HFF-1 cell line after 24 h of Hesperidin was 527 µM.Regarding these results, 10 µM AP, and 50 and 100 µM Hesperidin were selected for experimental concentrations.

The effect of AP and Hesperidin on ROS level
The intracellular ROS level was measured in PC3 and LNCap cell lines in response to AP (10 µM) and Hesperidin (50 and 100 µM) alone and combination groups (Fig. 4 (a and b)).As shown in Fig. 4 (a), the ROS level increased significantly after inoculation with 50 and 100 µM Hesperidin in the PC3 cell line in comparison to the untreated group.Furthermore, the ROS level significantly reduced after treating the PC3 cells with 10 µM AP with or without Hesperidin pretreatment.Figure 4 (b) presents the ROS level after inoculation with AP and Hesperidin in the LNCaP cell line.ROS level significantly increased after inoculation with 100 µM Hesperidin and the combination of 100 µM Hesperidin + 10 µM AP.However, 10 µM AP with or without 50 µM Hesperidin significantly reduced the ROS level.

The effect of AP and Hesperidin on P53 and P21 gene expression
As shown in Figs. 5 (a and c), P53 and P21 gene expression significantly increased after treatment with 50 and 100 µM Hesperidin with or without 10 µM AP compared to the untreated group in the PC3 cell line.Besides, 50 and 100 µM Hesperidin significantly increased the P53 and P21 gene expression in the LNCaP cell line (Fig. 5 (b and d)).

The effect of AP and Hesperidin on Survivin gene expression
As shown in Fig. 6 (c), 10 µM AP with or without the combination with 50 or 100 µM Hesperidin significantly decreased the Survivin gene expression in the PC3 cell line.Furthermore, Survivin gene expression significantly decreased after inoculation with 10 µM AP with or without 50 µM Hesperidin in the LNCaP cell line (Fig. 6 (d)).

The effect of AP and Hesperidin on Trypan blue apoptosis test
As presented in Fig. 7, 50 and 100 µM Hesperidin, 10 µM AP, and the combinations of 50 µM Hesperidin + 10 µM AP and 100 µM Hesperidin + 10 µM AP induce a significantly higher rate of apoptosis in both PC3 and LNCaP cell lines compared to the untreated group.

Discussion
As previously noted, prostate cancer is the second most frequent cancer in men (Bray et al. 2018).Conventional therapies for prostate cancer include chemotherapy, radiotherapy, and androgen deprivation therapy (anti-androgenic medications (flutamide and bicalutamide)), orchiectomy (in severe cases), and freezing of prostate tissues (Barani et al. 2020).However, this approached are envisioned with loss of libido, erectile dysfunction, bone mass loss, and obesity (Barani et al. 2020;Oun et al. 2018).In addition, the development of castration resistance, cancer recurrence, metastatic disease, and therapeutic regimen adverse effects are the main problems and have limited the treatment efficacy (Nakazawa et al. 2017;Katzenwadel and Wolf 2015).Hence, it's vital to find new potential therapy that has no toxic effects on healthy cells.In the current study, prostate cancer cell lines, PC3 and LNCaP were treated with AP (10 µM) and Hesperidin (50 and 100 µM) alone and in combination groups.To the best of our knowledge, this is the first study that is aimed to assess the synergism effects of AP and Hesperidin on cancer cells.Our results showed that the combinations of 50 µM Hesperidin + 10 µM AP and 100 µM Hesperidin + 10 µM AP induce a significantly higher rate of apoptosis in both PC3 and LNCaP cell lines.On contrary, Hesperidin did not affect the normal cell line (HFF-1).The IC50 of the normal cell Fig. 2 The resazurin assay shows the viability of the PC3 and LNCaP cell line after 24 h of Aprepitant.Aprepitant can cause prostate cancer cell death in both PC3 and LNCaP cell lines in a dose-and timedependent manner.Section (a): IC50 in PC3 cells treated with Aprepitant was 18.30 μM after 24 h.Section (b): IC50 in LNCaP cells treated with Aprepitant was 22.35 μM after 24 h Fig. 3 The resazurin assay shows the viability of the HFF-1 cell line after 24 h of Hesperidin.Hesperidin can cause HFF-1 cell death in a dose-and time-dependent manner with IC50 of 527 μM line (HFF-1) after treatment with Hesperidin was almost 2 times and 20 times higher than LnCaP and PC3 cell lines respectively.These results showed the safety of Hesperidin on the normal cell line, while it induces apoptosis in prostate cancer cells.
The mechanism underlying the synergism effect of AP and Hesperidin can be due to several molecular pathways.
ROS is one of the potential pathways, especially for AP.ROS formation during metabolism has been shown in different physiological functions (Moloney et al. 2018).In fact, the balance between ROS production and its scavenging with antioxidants has been properly maintained in healthy cells (Kim et al. 2016).However, cancer cells have dysregulated ROS hemostasis, leading to a higher ROS generation (Kim et al. 2016).A higher level of ROS has an antiapoptotic effect as a result of redox-sensitive transcription activation which includes nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) (Grivennikov and Karin 2010).NF-κB is located in the cytosol of healthy cells which is bonded to IκBα as inactive forms.Nevertheless, IκBα phosphorylation forms active NF-κB in cancer cells which can inhibit apoptosis and result in uncontrol cell growth (Wang et al. 1998).In fact, NF-κB can inhibit apoptosis by elevating anti-apoptotic genes including Bcl-2 and survivin (Arbab et al. 2012).Previous studies showed the overexpression of Bcl-2 in prostate cancer, B-cell lymphomas, colorectal cancer, and breast cancer (Kirkin et al. 2004).Therefore, reducing the ROS level in prostate cancer cells can lead to reduced antiapoptotic effects as a result of lower Bcl-2 and survivin gene expression.The results of the current study showed that the combination of 10 µM AP + 50 µM Hesperidin significantly reduced the ROS level in the PC3 cell line.Also, Bcl-2 and Survivin gene expression significantly decreased with the combination of 50 µM Hesperidin + 10 µM AP, and 100 µM Hesperidin + 10 µM AP in the PC3 cell line.Almost similar results were found in the LNCaP cell line.This mechanism can be one of the potential anti-cancer effects of Hesperidin and AP on prostatic cancer cells.On contrary, in a study done by Ning et al. on prostate cancer cells., it was shown that hesperidin can decrease cell growth and viability in a dose-dependent manner as a result of ROS elevation and MMP reduction (Ning et al. 2020).These results can be explained by the double sword feature of ROS, in which both lowering and elevating the ROS level in cancer cells can induce apoptosis and were identified to be potent therapeutic approaches in the cancer management (Javid et al. 2022b).
On the other hand, P53 and P21 pathways seem to be another potential anti-cancer mechanism, especially for Hesperidin on prostatic cancer cells.Various cell functions are regulated with P53 tumor suppressor factor including cell growth, invasion, and migration (Muller et al. 2011).In addition, P21 plays an important role in the cell-growth arrest, senescence, and suppression of the cell invasion (Abbas and  Lee et al. 2012).Its cell cycle arrest role can be due to the modulation of cell cycle regulatory proteins such as cyclins, cyclin-dependant kinases (CDK), and CDK inhibitors (Pandey and Khan 2021).In addition, it was reported that Hesperidin can increase P21 in various cancer cells including leukemia cell lines, colon cancer, breast cancer, and lung cancer (Oliveira et al. 2020).On the other hand, it was shown that AP can cause cell cycle arrest in G2/M and significantly decrease cyclin B1, as well as, increase P21 in other types of cancer cell lines (Obata et al. 2016).Besides, it was stated that AP can cause cancer cell death by regulating different pathways such as cell cycle-related genes (c-Myc, cyclin D1, cyclin B1, p21), P53, PI3K / Akt/ NF-kB, and apoptosis target genes (Bcl-2 and Bax) (Javid et al. 2022a).However, our results did not show significant changes in P53 and P21 in prostate cancer cells after treatment with 10 µM AP alone.
Furthermore, SP and its primary receptor, NK1R can be another pathway for the anti-cancer effect.As previously noted, SP's binding to NK1R can induce cancer cell progression, metastasis, and angiogenesis (Esteban et al. 2006).Further studies showed that prostate cancer cells overexpress NK1R (Cussenot et al. 1996).Based on the role of SP/ NK1R system in initiating and progression of cancer cells, it seems to be a potential target for anti-cancer treatments.Toward this end, Ebrahimi et al. stated SP/NK1R can induce proliferation and migration in prostate cancer cells by affecting apoptosis-related genes, cell cycle-related proteins, and increasing MMP-2 and MMP-9 expression (Ebrahimi et al. 1869).They showed Aprepitant can reverse these effects in both in vitro and in vivo experiments on prostate cancer cells (Ebrahimi et al. 1869).Although our results for the first time, showed the synergic anti-cancer effect of Hesperidin and AP on prostate cancer cells in both PC3 and LNCaP cell lines, this effect can be well explained by P53, P21, Bcl-2, Survivin, and ROS pathways only in the PC3 cell line.However, our results for these possible pathways of the anti-cancer effect of Hesperidin and AP were not straightforward in the LnCaP cell line.One possible explanation for these results can be the natural difference between the PC3 and LNCaP cell lines.For instance, a prior study showed Hesperidin can inhibit the testosterone-induced cell proliferation of the LNCaP cell line, while it had no effect on hormone-independent prostate cancer cells, PC3 (Lee et al. 2010).Therefore, the hormonal effects of these treatments can be an explanation for this difference and another potential pathway for their anti-cancer effects.In addition, the following limitations should be considered in this study.First, SP/NK1R pathway in prostate cancer cells was not fully investigated in the current study.Second, the signaling pathways of NF-κB were not evaluated.Finally, this is in vitro investigation and further in vivo studies are needed on this subject.
In conclusion, the current study showed the synergic anticancer effect of Hesperidin and Aprepitant on prostate cancer cells in both PC3 and LNCaP cell lines.The combination

Figure 6 (
Figure 6 (a and b) demonstrates the effect of AP and Hesperidin on Bcl-2 gene expression.Bcl-2 gene expression significantly decreased with 10 µM AP with or without 50 or 100 µM Hesperidin pretreatment in the PC3 cell line.Besides, 10 µM

Fig. 4
Fig. 4 The effect of Hesperidin and Aprepitant on ROS level in PC3 and LNCaP cell lines.Hesperidin significantly increased the ROS level.While Aprepitant significantly reduced the ROS level.Section (a): Aprepitant with or without 50 and 100 μM Hesperidin significantly reduced the ROS level in the PC3 cell line.Section (b): Aprepitant with or without 50 μM Hesperidin significantly decreased ROS

Fig. 5
Fig. 5 The effect of Hesperidin and Aprepitant on P53 and P21 gene expression in PC3 and LNCaP cell lines.Section (a): 50 and 100 μM Hesperidin with or without 10 μM Aprepitant significantly increased P53 gene expression in the PC3 cell line.Section (b): 50 and 100 μM Hesperidin significantly increased P53 gene expression in the LNCaP cell line.Section (c): 50 and 100 μM Hesperidin with or without 10 μM Aprepitant significantly

Fig. 6
Fig. 6 The effect of Hesperidin and Aprepitant on Bcl-2 and Survivin gene expression in PC3 and LNCaP cell lines.Section (a): 10 μM Aprepitant with or without 50 and 100 μM Hesperidin significantly reduced Bcl-2 gene expression in the PC3 cell line.Section (b): 10 μM Aprepitant with or without 50 and 100 μM Hesperidin significantly reduced Bcl-2 gene expression in the LNCaP cell line.Section (c): 10 μM Aprepitant with or without 50 and 100 μM Hesperidin significantly reduced Survivin gene expression in the PC3