Picropodophyllin Inhibits the Proliferation of Human Prostate Cancer DU145 and LNCaP Cells via ROS Production and PI3K/AKT Pathway Inhibition

Abstract

The reactive oxygen species (ROS) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway play critical roles in the pathogenesis of prostate cancer by modulating cell proliferation. Picropodophyllin (PPP), an inhibitor of the insulin-like growth factor 1 receptor (IGF-1R), exerts significant antitumor effects via the PI3K/AKT signaling pathway. However, the effects of PPP on prostate cancer via ROS production and the PI3K/AKT signaling pathway remain elusive. Herein, we focused on examining the antitumor effects of PPP on DU145 and LNCaP human prostate cancer cells to determine the possible molecular mechanism. Our data indicated that the inhibitory effect of PPP on the proliferation of DU145 and LNCaP human prostate cancer cells was mediated by apoptosis induction and cell cycle blockade. Furthermore, PPP significantly influenced the expression of apoptosis-related, cell cycle, ROS production, and PI3K/AKT signaling proteins. These findings suggest that PPP can induce cell cycle arrest and apoptosis via the production of ROS and inhibition of PI3K/AKT signaling pathway, thereby suppressing the proliferation of prostate cancer cells.

INTRODUCTION

Prostate cancer is a common malignancy in humans. Approximately 1.3 million new cases of prostate cancer and 359000 associated deaths were recorded worldwide in 2018, according to the estimates of GLOBOCAN.¹⁾ The major risk factors for prostate cancer include age, race, and heritability.^2–4) Despite improved treatment modalities, the treatment of prostate cancer remains unsatisfactory. Androgen deprivation therapy (ADT) is the main treatment option for advanced prostate cancer.⁵⁾ ADT induces a series of adverse reactions, leading to obesity, metabolic syndrome, osteoporosis, sarcopenia, diabetes mellitus, cardiovascular disease, gynecomastia, and sexual dysfunction.^5–7) Therefore, it is particularly important to develop safe and effective therapeutic strategies for prostate cancer.

Reactive oxygen species (ROS) are a group of reactive short-lived oxygen-containing species that include superoxide, singlet oxygen, hydrogen peroxide, hydroxyl radicals, and peroxyl radicals. ROS play crucial roles in malignancies, and a substantial increase in ROS levels can initiate cell apoptosis, damage, and senescence.⁸⁾ ROS can interplay with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, whose downstream signaling pathway is associated with cell proliferation, apoptosis, and oncogenesis.⁹⁾ Auriculasin decreases prostate cancer growth via ROS-mediated caspase-independent pathways and suppresses the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling pathway.¹⁰⁾

Picropodophyllin (PPP) is derived from the rhizome of a Berberidaceae family member, Dysosma versipellis (Hance.) M. Cheng; it exhibits various pharmacological activities, including anti-inflammatory, anti-aging, and antitumor activities.^11–13) Zhang et al. revealed that PPP considerably reduced the lung tumor multiplicity and tumor load.¹⁴⁾ PPP also lowered tumor volume and weight in an in vivo xenograft model and caused a dose-dependent decrease in SKOV-3 cell survival in vitro.¹⁵⁾ Moreover, PPP attenuates the proliferation and survival of diffuse large B-cell lymphoma cells.¹⁶⁾ According to E et al., PPP selectively suppresses the growth of human hepatocellular carcinoma cells by triggering the caspase-dependent mitochondrial pathway cell death mechanism, with no cytotoxicity detected in normal cells.¹⁷⁾

PPP can be used as an inhibitor of insulin-like growth factor 1 receptor (IGF-1R).^18,19) It specifically attenuates IGF-1R activity by suppressing IGF-1R phosphorylation and downstream signaling cascades.²⁰⁾ PPP triggers a decrease in IGF-1R phosphorylation and attenuates cell proliferation via the PI3K/AKT signaling pathway.¹³⁾ Interestingly, IGF-1R can activate the PI3K/AKT signaling pathway and influence cell proliferation and apoptosis.^21,22) PPP can also inhibit the growth and proliferation of human endometrial cancer cells by blocking the IGF-1R/PI3K/AKT signaling pathway.⁸⁾ In the present study, we focused on the suppressive effect of PPP on the proliferation of prostate cancer cells, and further explored whether this occurs via the ROS and PI3K/AKT signaling pathway.

MATERIALS AND METHODS

Materials

PPP (purity ≥98%) was purchased from Herbest Technology (Shanghai, China). Fetal bovine serum, minimum essential medium (MEM), 1% MEM non-essential amino acids, and 1% glutamate additives were purchased from Gibco (Grand Island, NY, U.S.A.). Phenylmethylsulfonyl fluoride (PMSF), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), radioimmunoprecipitation assay (RIPA) lysis buffer, protease and phosphatase inhibitors, 1% penicillin mixture, Hoechst 33258 dye, Annexin V apoptosis detection kit with propidium iodide (PI), cell cycle detection kit, bicinchoninic acid (BCA) protein assay kit, 4% tissue cell fixation solution, 1% crystal violet dye, 0.25% trypsin containing ethylene diamine tetraacetic acid (EDTA), and reactive oxygen species assay kit were obtained from Solarbio Technology Co., Ltd. (Beijing, China). The cleaved caspase-9 (Cat. No. ab2324; 1 : 1000), cyclin-dependent kinase 1 (CDK1; Cat. No. ab133327; 1 : 50000), Cyclin B1 (Cat. No. ab32053; 1 : 50000), and P-Y607-p85α (Cat. No. ab182651; 1 : 1000) antibodies were purchased from Abcam (Cambridge, U.K.). β-Actin (Cat. No. TA-09; 1 : 2000) and peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) (H + L) (Cat. No. ZB-2305; 1 : 50000) antibodies were purchased from Zhongshan Jinqiao Biological Co., Ltd. (Beijing, China). p-AKT (Cat. No. 66444-1-Ig; 1 : 2000), caspase 9 (Cat. No. 10380-1-AP; 1 : 2000), and p85α (Cat. No. 60225-1-Ig; 1 : 20000) antibodies were purchased from Proteintech Group (Hubei, China). Poly(ADP-ribose) polymerase (PARP; Cat. No. 9542, 1 : 1000), P-CDK 1 (Cat No. 4539; 1 : 1000), and AKT (Cat. No. 9272 s; 1 : 1000) antibodies were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.).

Cell Culture

Human prostate cancer DU145 and LNCaP cells were purchased from the Chinese Academy of Sciences (Shanghai, China). DU145 cells were kept in an incubator (humidified condition and 5% CO₂) at 37 °C in MEM medium (Cat. No. 8120263; Gibco) containing 10% fetal bovine serum (Cat. No. 10091-148; Gibco), 1% penicillin mixture (Cat. No. P1400; Solarbio), 1% MEM non-essential amino acids (Cat. No. 11140-050; Gibco), 1% glutamate additive (Cat. No. 35050061; Gibco), and 1% sodium pyruvate (Cat. No. SP0100; Solarbio). LNCaP cells were kept in an incubator (humidified condition and 5% CO₂) at 37 °C in Roswell Park Memorial Institute-1640 medium (Cat. No. R8758; Sigma, St. Louis, MO, U.S.A.), containing 10% fetal bovine serum (Cat. No. 10091-148; Gibco) and 1% penicillin mixture (Cat. No. P1400; Solarbio, Beijing, China).

MTT Assay

Cell proliferative activity was examined using the MTT assay. The cells (8 × 10³/well) were seeded in a 96-well plate and exposed to PPP at concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 µM. After 24 h of treatment, the MTT solution (20 µL) was added to each well and incubated for 120 min before analysis. All results were obtained from at least three independent experiments. Absorbance was measured using a microplate reader (Infinite 200 PRO, Tecan, Austria) at 490 nm. The inhibition rate was calculated using GraphPad Prism 5.0 software (San Diego, CA, U.S.A.) for statistical analysis.

Colony Formation Assay

The digestion of DU145 and LNCaP cells (in logarithmic growth phase) was carried out using trypsin solution (0.25%) containing EDTA (Cat. No. 71300; Solarbio), followed by cell seeding (200 cells/well) in a 6-well plate. The cells were then exposed to various PPP concentrations for 24 h. On the second day, fresh medium was added, and the cells were cultured in the incubator for the next 15 d. Fixative solution (4%) (Cat. No. P1110; Solarbio) was used for fixing colonies (for 15 min), followed by staining with 1% crystal violet dye (Cat. No. G1062; Solarbio) for 15 min. After washing with the phosphate buffer, images were recorded using a camera. All results were obtained from at least three independent experiments. The number of colonies formed was counted using the ImageJ software (NIH, Bethesda, MD, U.S.A.) and the colony formation rate was calculated using GraphPad Prism 5.0 software for statistical analysis.

Hoechst 33258 Staining

The digestion of DU145 and LNCaP cells (in logarithmic growth phase) was carried out using a trypsin solution (0.25%) containing EDTA (Cat. No. 71300; Solarbio), followed by seeding (1.1 × 10⁵ cells/well) in a 6-well plate. The cells were exposed to various PPP concentrations for 24 h. The cells were fixed using a fixation solution (4%) for 20 min and stained with Hoechst 33258 solution (Cat. No. C0003; Beyotime, China). A fluorescence microscope (DMI3000B; Leica, Wetzlar, Germany) was used to capture the images.

Apoptotic Assay

Cells (1.1 × 10⁵ cells/well) were seeded in a 6-well plate and exposed to various concentrations of PPP (0.6, 0.8, and 1.0 µM) for 24 h, followed by cell collection. Next, cells were washed twice with phosphate-buffered saline (PBS) and resuspended in a binding buffer (400 µL). Annexin V-fluorescein isothiocyanate (FITC) and PI solution (5 µL) was added to the sample, which was then incubated for 15 min at 25 °C before performing flow cytometry (FACSCanto II; Becton, Dickinson and Company, NJ, U.S.A.). All results were obtained from at least three independent experiments. Finally, the apoptotic rate was analyzed using GraphPad Prism 5.0 software for statistical analysis.

Evaluation of Cell Cycle

Cells (1.1 × 10⁵cells/well) were cultured in a 6-well plate to evaluate the cell cycle. Fresh medium was added to these cells at various PPP concentrations for 24 h. Cells were then collected and cooled ethanol (70%) was used for cell fixation at 4 °C for 2–4 h. After washing thrice with PBS, ribonuclease (RNase) A solution was added to the cells and incubated at 37 °C for 30 min. Next, PI staining solution was added at 37 °C in the dark. The cells were then evaluated using flow cytometry (FACSCanto II; Becton, Dickinson and Company). All results were obtained from at least three independent experiments. Finally, cell distribution in each period was analyzed using GraphPad Prism 5.0 software for statistical analysis.

Detection of ROS

Cells (1.1 × 10⁵ cells/mL) were cultured in a 6-well plate for 24 h. Fresh medium (with various PPP concentrations) was added for 24 h, followed by cell collection and suspension in 2,7-dichlorofluorescin diacetate (DCFH-DA) solution (Cat. No. CA1410; Solarbio) at 37 °C for 20 min. The cells were then washed and suspended in PBS. Flow cytometry (FACSCanto II; Becton, Dickinson and Company) was used to analyze the cells. A fluorescence microscope (DMI3000B; Leica, Leica Microsystems CMS GmbH) was used to capture the images. All results were obtained from at least three independent experiments. Finally, data obtained from flow cytometry were statistically analyzed using GraphPad Prism 5.0 software.

Quantitative Real-Time PCR

Total RNA of the cells was isolated by Trizol reagents. Then the reverse transcription was performed by using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). SYBR^® Premix Ex Taq II (TaKaRa, Dalian, China) was used to analyze the mRNA expression of selected genes.

Western Blotting

Cells (2.2 × 10⁵ cells/mL) were incubated with PPP at 600, 800, and 1000 nM at 37 °C for 24 h, followed by cell collection. RIPA lysis buffer containing PMSF (Cat. No. P0100; Solarbio) and phosphatase inhibitors (Cat. No. P12600; Solarbio) was used to extract the cells for 0.5 h. Lysates were centrifuged at 12000 × g at 4 °C for 15 min. A BCA protein assay kit was used to determine the concentration of proteins. The protein lysates were diluted to equal concentrations, followed by denaturation for 5 min at 100 °C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%) was carried out to separate the total protein extracts (in equal amounts), which were then transferred (electrically) onto polyvinylidene fluoride membranes. For blocking purposes, skimmed milk (5%) was used in a Tris-buffered saline and Tween-20 mixture for 60 min at room temperature. Various specific primary and secondary antibodies (conjugated with HRP) were used to detect the proteins on the membranes. The SuperSignal West Femto Chemiluminescent Substrate Kit (Cat. No. 34095; Thermo Scientific) was used for chemiluminescence detection. ImageJ software (NIH) was used for quantitative analysis. β-Actin was used as an internal control to standardize the obtained data. Each experimental procedure was independently performed three times. Finally, the data were analyzed using GraphPad Prism 5.0 software for statistical analysis.

Statistical Analysis

Results were obtained from at least three independent experiments and indicated as the mean ± standard deviation. GraphPad Prism 5.0 was used to determine the statistical variations between two groups using the Student’s t-test. p < 0.05 was considered to be statistically significant.

RESULTS

PPP Decreases the Viability and Inhibits the Proliferation of Human Prostate Cancer DU145 and LNCaP Cells

To evaluate the impact of PPP on the viability of human prostate cancer cells, the viability of DU145 and LNCaP cells were evaluated using the MTT assay after exposure to various concentrations of PPP (0, 0.2, 0.4, 0.6, 0.8, and 1.0 µM). PPP significantly attenuated the viability of DU145 and LNCaP cells, with IC₅₀ values of 0.802 and 0.899 µM, respectively (Fig. 1A). As the concentration of PPP increased, DU145 and LNCaP cells became smaller and their number was reduced. Adherence and transparency of DU145 and LNCaP cells decreased after PPP treatment (Fig. 1B). In addition, the colony formation abilities of DU145 and LNCaP cells were evaluated to validate the proliferation inhibitory effect of PPP. It was observed that the number of clones diminished and became smaller upon PPP treatment (Fig. 1C).

Fig. 1. Picropodophyllin (PPP) Decreases the Viability and Inhibits the Proliferation of Human Prostate Cancer DU145 and LNCaP Cells

(A) DU145 and LNCaP cells were treated with PPP (0, 0.2, 0.4, 0.6, 0.8, and 1.0 µM) for 24 h. The 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to determine the cell viability of both cell lines. (B) Inverted phase contrast microscope was used to observe the morphological changes in PPP-treated DU145 and LNCaP cells. (C) DU145 and LNCaP cells were treated with PPP (0.6, 0.8, and 1.0 µM) for 24 h. Colony formation assay was used to evaluate the proliferation of PPP-treated DU145 and LNCaP cells. Data are presented as the mean ± standard deviation (S.D.) (n = 3). * p < 0.05 and ** p < 0.01 compared to the control group.

PPP Induces the Apoptosis of Human Prostate Cancer DU145 and LNCaP Cells

To determine the effect of PPP on the apoptotic process of the DU145 and LNCaP cells, cell incubation was carried out in the various concentration of PPP (0.6, 0.8, and 1.0 µM) for 24 h. Hoechst 33258 staining was used to assess PPP-induced apoptosis in both the cell lines. After PPP treatment, DU145 and LNCaP cells showed a pathological morphology equivalent to that of the apoptotic process, such as condensation of chromatin and nuclei with dense fluorescence and bright white emission. No morphological signs of apoptosis were observed in untreated cells (Fig. 2A). Annexin V-FITC and PI double staining was used to further confirm the impact of apoptosis induction by PPP. Treatment with PPP (0.6, 0.8, and 1.0 µM) markedly induced apoptosis in DU145 and LNCaP cells after 24 h. The percentage of apoptotic DU145 cells increased from 6.86 to 8.90% (0.6 µM), 12.36% (0.8 µM), and 18.33% (1.0 µM), and the percentage of apoptotic LNCaP cells increased from 4.90 to 9.30% (0.6 µM), 10.30% (0.8 µM), and 13.10% (1.0 µM) (Fig. 2B).

Fig. 2. PPP Induces the Apoptosis of Human Prostate Cancer DU145 and LNCaP Cells

(A) DU145 and LNCaP cells were treated with PPP (0.6, 0.8, and 1.0 µM) for 24 h. Hoechst 33258 staining was performed to observe the nuclear change in DU145 and LNCaP cells after PPP treatment for 24 h. (B) Apoptosis in PPP-treated DU145 and LNCaP cells was observed via Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining using flow cytometry. (C) Expression levels of caspase-9, cleaved caspase-9, poly(ADP-ribose) polymerase (PARP), and cleaved PARP in PPP-treated DU145 and LNCaP cells were determined using Western blotting. Data are presented as the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 compared to the control group.

Western blotting was used to detect the expression of proteins involved in the mitochondrial apoptosis pathway to investigate the underlying mechanism of PPP in apoptosis. The results showed that the ratios of cleaved caspase-9/caspase-9 and cleaved PARP/PARP were remarkably increased after PPP treatment compared to that of the control group in DU145 and LNCaP cells (Fig. 2C).

PPP Induces Cell Cycle Arrest in DU145 and LNCaP Cells

We performed flow cytometry to identify the impact of PPP on cell cycle progression. The results revealed that DU145 and LNCaP cells were both significantly arrested in the G2/M phase, and the number of these cells was significantly decreased in the G0/G1 phase (Fig. 3A). However, the distribution of both cell lines did not change significantly during S phase. To further characterize the contribution of PPP to cell cycle regulation, Western blotting was performed to evaluate the expression levels of the related proteins. As results analyzed by flow cytometry (FACSCanto II, Becton, Dickinson and Company) showed that both cell lines were augmented in G2/M phase and the numbers of both cell lines were decreased by PPP in G0/G1 phase, we detected the expression levels of G2/M phase regulatory proteins. The results showed that the ratio of G2/M phase regulatory proteins, p-CDK1/CDK1, was reduced by PPP. However, cyclin B1 expression was considerably elevated after PPP treatment (Fig. 3B).

Fig. 3. PPP Induces Cell Cycle Arrest in DU145 and LNCaP Cells

(A) Quantification analysis of the cell cycle distribution in DU145 and LNCaP cells was done using flow cytometry after treatment with PPP (0.6, 0.8, and 1.0 µM) for 24 h. (B) Expression levels of cyclin-dependent kinase 1 (CDK1) and Cyclin B1 in DU145 cells treated with PPP (0.6, 0.8, and 1.0 µM) were determined using Western blotting. Quantitative analysis was performed to analyze the expression levels of CDK1 and Cyclin B1 in DU145 and LNCaP cells treated with PPP (0.6, 0.8, and 1.0 µM). Data are presented as the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 compared to the control group.

PPP Induces Apoptosis and Cell Cycle Arrest in DU145 and LNCaP Cells via ROS Generation and PI3K/AKT Pathway Inhibition

ROS was reported to regulate cell proliferation and apoptosis.^23,24) We assumed that the apoptosis and cell cycle arrest of PPP on prostate cells might be associated with ROS. To evaluate the variations in ROS levels after incubation with PPP, ROS content was determined after staining with the fluorescent probe DCFH-DA. As the DU145 and LNCaP cells were exposed to PPP, both DCFH-DA fluorescence intensities were considerably elevated compared to those in the control group, as depicted in Figs. 4A and B. We then performed dihydroethidium staining, a superoxide anion fluorescent probe, to evaluate the change in the superoxide anion fluorescent content. The results showed that red fluorescence intensity increased after PPP treatment (Fig. 4C).

Fig. 4. PPP Induces Apoptosis and Cell Cycle Arrest in DU145 and LNCaP Cells via Reactive Oxygen Species (ROS) Generation and the Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B (AKT) Signaling Pathway Inhibition

(A) Total cellular ROS levels were determined via 2′,7′-dichlorofluorescin diacetate (DCFH-DA) staining using a fluorescence microscope. (B) Quantitative analysis of total cellular ROS levels. (C) Levels of superoxide anion detected via DHE staining using a fluorescence microscope. (D) Expression level of ROS related enzymes genes upon PPP treatment. (E) Expression levels of PI3K and AKT in DU145 and LNCaP cells treated with PPP (0.6, 0.8, and 1.0 µM). Data are presented as the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 compared to the control group.

To elucidate the possible mechanism by which PPP increases ROS levels, the expression levels of genes encoding ROS-scavenging enzymes, including catalase (CAT) and glutathione peroxidase 1 (GPX-1), and ROS-generating enzymes, including nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX1 and NPX3) and cytochrome b-245 beta chain (CYBB), were determined using real-time PCR, with glyceraldehyde-3-phosphate dehydrogenase as the reference gene. As shown in Fig. 4D, after treatment with PPP (1.0 µM), there were no differences in the expression levels of ROS-scavenging enzymes, while those of ROS-generating enzymes (NOX1 in DU145 cells, and NOX1 and NOX3 in LNCaP cells) were significantly increased. Therefore, PPP increased ROS levels by affecting ROS-generating enzymes rather than ROS-scavenging enzymes.

PPP was found to attenuate cell proliferation via the PI3K/AKT signaling pathway,¹³⁾ and this signaling pathway was also related to ROS.⁸⁾ Therefore, we detected variations in the expression levels of proteins involved in the PI3K/AKT signaling pathway. We found that the ratios of P-Y607-p85α/p85α and p-AKT/AKT decreased considerably in both cell lines after treatment with PPP (Fig. 4E).

DISCUSSION

In modern medicine, cancer can be classified as a chronic non-communicable disease, which is recrudescence and exhibits difficulty in curing.²⁵⁾ Cancer is a major public issue worldwide, with considerable mortality and morbidity despite advances in diagnosis and treatment technologies. Apoptosis is a major suicide mechanism important for maintaining cellular homeostasis. It is triggered by the stimulation of pre-existing cascades in each cell in response to death.²⁶⁾ In normal cells, the apoptotic process helps to maintain a balance between cell proliferation and cell death.²⁷⁾ On the other hand, many strategies have been developed in cancerous cells to resist death, including DNA mutations in pro-apoptotic protein-coding genes, elevated expression of anti-apoptotic proteins and/or pro-survival signals, and more recently, pro-apoptotic gene silencing mediated by DNA hypermethylation.²⁸⁾ The major process that leads to cellular proliferation is the cell cycle. Cell proliferation and death are inextricably associated. Wang et al. revealed that apoptosis (induced via paclitaxel) might occur after mitotic arrest or abnormal mitotic exit into G1-like “multinucleate state.”²⁹⁾ Whether apoptosis occurs after cell cycle arrest or in the process of the cell cycle, cell proliferation stops once cell cycle arrest occurs.

Currently, cytotoxic drugs are still the basic principles of antitumor drug design. Cytotoxic drugs not only induce cancer cell death but also damage normal cells. Therefore, it is necessary to find drugs with high efficiency and low toxicity for cancer treatment. Traditional Chinese medicine has been practiced for thousands of years and is now widely regarded as a cancer therapy option.³⁰⁾ Traditional Chinese medicine is widely acknowledged as a mainstream form of complementary and alternative therapy with therapeutic effects for people with cancer in China, owing to its efficiency and fewer side effects.³¹⁾

In recent studies, it has been demonstrated that an elevated level of ROS plays a key role in triggering the cellular apoptotic process.^32,33) Fan et al. revealed that Bruceine D triggered lung cancer cell death and autophagy via the mitogen-activated protein kinase (MAPK) signaling cascade (mediated by ROS) in vitro and in vivo.³⁴⁾ According to Xue et al., ROS increases the cytotoxicity of cisplatin by triggering the apoptotic process and autophagy in tongue squamous cell carcinoma cells.³⁵⁾ Photodynamic treatment (linked with berberine) induces autophagy and death in renal cancer cells through ROS, according to a previous study.³⁶⁾ The level of cellular ROS increased following PPP therapy, according to our findings. PPP also increases the rate of apoptosis in prostate cancer cells. We speculated that the elevation in ROS levels is the cause of apoptosis in cells exposed to PPP. Elevation of either oxygen pressure or ROS levels has been revealed to cause mutations, chromosomal and DNA damage, attenuation of cell division, and tumor advancement.³⁷⁾ In human esophageal cancer cells, Kwak et al. indicated that 3-deoxysappanchalcone promotes cell cycle arrest (triggered by ROS) via the c-jun N-terminal kinase/p38 mitogen-activated protein kinase signaling cascade.³⁸⁾ A previous study reported that calycosin promotes cell cycle arrest in the G0/G1 phase by modulating MAPK, signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappa-B (NF-κB) signaling cascades mediates via ROS.³⁹⁾ In human hepatocellular carcinoma, pogostemon cablin resulted in ROS-stimulated DNA damage, leading to cell cycle arrest in vitro and in vivo.⁴⁰⁾ Our results showed that cell cycles were markedly arrested in the G2/M phase, while the cellular ROS levels were elevated after treatment with PPP. We inferred that ROS may trigger checkpoint responses via induced mutations or DNA damage, affecting the distribution of the cell cycle.

ROS are thought to play an important role as second messengers in triggering the PI3K/AKT signaling pathway.⁴¹⁾ In TM3 Leydig cells, Zhu et al. revealed that ROS buildup leads to abamectin-induced apoptosis and autophagy via inactivation of the PI3K/AKT/mTOR cascade.⁴²⁾ Metformin promotes the susceptibility of colorectal cancer cells to cisplatin via the PI3K/AKT signaling pathway (mediated by ROS), according to a study by Zhang.⁴³⁾ Dexamethasone causes osteoblast apoptosis via the PI3K/AKT/glycogen synthase kinase 3β signaling cascade mediated by ROS, according to Deng et al.⁴⁴⁾ Herein, we found that PPP can cause excessive ROS generation in DU145 and LNCaP cells, resulting in oxidative stress. After PPP treatment, the expression of p-PI3K and p-AKT was considerably reduced. PPP enhances PI3K and AKT expression The results revealed the triggering of the PI3K/AKT signaling pathway. Our findings suggest that excessive ROS accumulation may be related to the PI3K/AKT signaling pathway. The PI3K/AKT cascade appears to be involved in the development of many malignancies and governs a variety of cellular activities, including differentiation, proliferation, metastasis, and metabolism.⁴⁵⁾ We speculated that ROS generation and PI3K/AKT pathway inhibition contributed to PPP-induced apoptosis and cell cycle arrest in DU145 and LNCaP cells, but the relationship between ROS production and PI3K/AKT pathway inhibition needs further exploration.

To the best of our knowledge, this is the first study to demonstrate that PPP exerts antitumor effects on human prostate cancer. However, it should be noted that the expression levels of CDK1 and Cyclin B1 were different from those reported in some studies. The expression of these two proteins were reported to be time-dependent when cell cycle arrest occurred in the G2/M phase.⁴⁶⁾ That study reported that the expression levels of CDK1 were reduced, whereas those of Cyclin B1 were increased when the cells were treated with physapubescin B for 0, 6, 12, 18, and 24 h.

In summary, our data demonstrate that PPP can cause apoptosis and cell cycle arrest in DU145 and LNCaP human prostate cancer cells via ROS production and PI3K/AKT pathway inhibition. This study elucidates a previously undiscovered mechanism for PPP-induced apoptosis and cell cycle arrest, providing evidence that PPP can be used to treat or prevent human prostate cancer. However, further research is necessary to determine the binding sites and confirm the antitumor efficacy of PPP in vitro.

Acknowledgments

We would like to thank the National Natural Science Foundation of China (Grant No. 31471338 to Qiusheng Zheng), Taishan Scholars Construction Engineering of Shandong Province (to Defang Li), the Yantai High-End Talent Introduction Plan “Double Hundred” (to Defang Li), the Introduction and Cultivation Project for Young Creative Talents of Higher Education of Shandong Province (to Guoli Wang), and the Introduction and Cultivation Project for Young Creative Talents of Higher Education of Shandong Province (to Minjing Li) for their support.

Conflict of Interest

The authors declare no conflict of interest.

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