Eprinomectin, a novel semi-synthetic macrocylic lactone is cytotoxic to PC3 metastatic prostate cancer cells via inducing apoptosis

Abstract

Prostate Cancer (PCa) is the second most common cancer among men in United States after skin cancer. Conventional chemotherapeutic drugs available for PCa treatment are limited due to toxicity and resistance issues. Therefore, there is an urgent need to develop more effective treatment for advanced PCa. In this current study, we focused on evaluating the anti-cancer efficacy of Eprinomectin (EP), a novel avermectin analog against PC3 metastatic PCa cells. EP displayed robust inhibition of cell viability of PC3 cells in addition to suppressing the colony formation and wound healing capabilities. Our study showed that EP targets PC3 cells via inducing ROS and apoptosis activation. EP treatment enforces cell cycle arrest at G0/G1 phase via targeting cyclin-dependent kinase 4 (CDK4) and subsequent induction of apoptosis in PC3 cells. At the molecular level, EP effectively inhibited the expression of various cancer stem cell markers such as ALDH1, Sox-2, Nanog, Oct3/4 and CD44. Interestingly, EP also inhibited the activity of alkaline phosphatase, a maker of pluripotent stem cells. Of note, EP treatment resulted in the translocation of ß-catenin from the nucleus to the cytoplasm indicating that EP antagonizes Wnt/ß-catenin signaling pathway. Western blotting analysis revealed that EP downregulated the expression of key cell cycle markers such as cyclin D1, cyclin D3, CDK4 and c-Myc while also inhibiting anti-apoptotic markers such as Mcl-1, XIAP, c-IAP1 and survivin in PC3 cells. On the other hand, EP treatment resulted in the activation of pH2A.X, Bad, caspase-9, caspase-3 and cleavage of PARP1. Taken together, our data suggests that EP is a potential agent to treat advanced PCa cells via modulating apoptosis signaling.

1. Introduction:

Prostate cancer (PCa) is one of the leading causes of cancer death among men in United States¹. About 1 in 39 men will die of PCa every year. Men above the age of 50 have high risk of developing PCa. According to the American Cancer Society’s estimation, in 2020 there would be 191,930 cases of PCa and about 33,330 deaths from PCa. Current available treatments for PCa include surgery, radiation therapy, chemotherapy, hormonal therapy or a combination of these. Chemotherapy is the most commonly used treatment strategy for metastatic PCa. There are several chemotherapeutic drugs available for treating PCa including taxol derivatives, mitoxantrone, epothilones and abiraterone. Several studies have shown the effectiveness of these drugs in phase II and phase III clinical trials in PCa patients^2–5. However, chemotherapy also has adverse side effects on patients⁶. Hormonal therapy or Androgen Deprivation Therapy (ADT) is the most successful therapy so far for PCa. The hormones such as androgens, testosterone and dihydrotestosterone show critical involvement in PCa⁷. However, after 18 to 24 months of treatment the patients develop resistance towards this therapy and develop Castration Resistant Prostate Cancer (CRPC)⁸. The CRPC show the ability to metastasize to distant organs like lymph nodes and bones. Thus, there appears to be an unmet need for developing new therapies for advanced PCa with very minimal toxicities.

In the last two decades, anti-parasitic drugs have been identified to be potential anti-cancer agents. For example, Artemisinin which is used for treating malaria since 1973, is most effective for falciparum malaria and cerebral malaria clinically⁹. Artemisinin and its derivatives have been reported to inhibit the growth of various cancers such as leukemia, melanoma, prostate cancer, kidney cancer and breast cancer⁹. Chloroquine is another antimalarial drug which has long been used to treat or prevent malaria. Chloroquine and hydroxychloroquine are identified as potent anti-cancer agents¹⁰. Several clinical trials are being conducted which emphasize the effects of chloroquine as a novel anti-tumor drug¹¹. Chloroquine has been used in clinical trials in combination with conventional chemotherapeutic drugs for cancers¹². In line with other anti-parasitic drugs, Niclosamide is another oral anti-helminthic drug used to treat tapeworm infection. It has been in use for humans for about 50 years¹³. Recently, several groups have independently identified Niclosamide as a potent anti-cancer agent^14–16. Evidence show that Niclosamide targets multiple pathways in cancer such as wnt/β-catenin, notch and NF-kB, most of which are closely associated with cancer stem cells (CSCs)¹⁷.

Avermectins are a class of drugs which have wide application as pesticides and anti-parasitic agent for humans and animals. They are macrolytic lactones produced by the fungus Streptomyces avermitilis. Drugs of avermectin class which are primarily used against parasitic diseases in animals and humans have been identified to have anti-cancer potential against various cancers^18,19. The lactone derivatives selamectin and ivermectin have been identified as candidate compounds to block SIN3-PAH2 interaction in breast cancer cells¹⁸. Some classes of avermectins such as Abamectin, Emamectin and Ivermectin act as inhibitors of MDR1 receptors in neuroblastoma cell lines. Among these three drugs, Ivermectin is shown to have more affinity towards MDR1 followed by Emamectin and Abamectin¹⁹. Eprinomectin (EP) is a novel macrocyclic lactone belonging to the avermectin class of drugs, which has excellent broad spectrum anti-parasitic effect when applied topically in cattles⁵³. Despite its greater potency and structural similarity with avermectins, the anti-cancer potential of EP has not been evaluated to date. In this study, we have evaluated the anti-cancer effects of EP on metastatic PC3 prostate cancer cells using various cellular assays. We have also elucidated the potential anti-cancer mechanisms by which EP could target metastatic PCa.

2. Materials and methods

2.1. Prostate cancer cell lines

PCa cell lines (PC3, DU145, LNCaP, VCaP, C4–2 and 22RV1) used in this study were purchased from ATCC. In addition, benign prostate cell line RWPE-1 was used as a control. These PCa cell lines cells were cultured in RPMI-1640 (Lonza) media supplemented with 10% Fetal Bovine Serum (FBS) from Hyclone, 1.5% antimycotic from Sigma, and 1% Gentamycin obtained from MP Biotech at 37°C with 5% CO₂ incubator. RWPE-1 was cultured in KSFM media (Gibco) supplemented with BPE (Bovine Pituitary Extract - 25μg/ml) and EGFR (Epidermal Growth Factor Receptor – 5ng/ml).

2.2. Drug

Eprinomectin (Sigma), a derivative of avermectin class of drugs with a molecular weight of 914 g/mol was used in this study. It was dissolved in Dimethyl Sulfoxide (DMSO) purchased from Fisher Scientific to make 10mM stock. Further dilutions of EP were made in plain RPMI media and stored in −20°C until further use.

2.3. MTT cell viability assay

To study the effects of EP on cell viability, PC3 cells were treated with different concentrations of EP and cell viability was assessed using MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) according to previously described protocols^20,21. PCa cells were seeded in 96 well plates with approximately 3000 cells per well with 100μl of complete media including antibiotics. Plates were incubated in 37°C with 5% CO₂ until the cells were 60% confluent. The cells were treated with varying concentrations of EP such as 5μM, 10μM, 25μM, 50μM and 100μM. After 48 hours of drug treatment, 5mg/ml of MTT dye was added to each well and incubated for three hours. After incubation period, 150μl of MTT solubilizing agent containing 90% iso-propanol and 10% Triton-X was added to each well. Plates were kept in shaker for about 15 minutes until all the crystals were solubilized. After this period of incubation, the plates were read at an absorbance of 590nm. The % cell viability was calculated using OD of experimental cells / OD of control cells × 100.

2.4. Colony formation assay

The effect of EP on clonogenic survival ability of PC3 cells was studied using colony formation assay. Colony formation assay was performed as per Dasari., et al with few modifications²². Approximately 500 cells were cultured in a six well plate in complete media and incubated in 37°C with 5% CO_2. After 3 to 4 hours, when the cells attach to the plate, they were treated with different concentrations of EP (5μM, 10μM, 25μM, 50μM and 100μM). The plates were incubated for two weeks. Following incubation, the media was removed and 2ml of fixing agent per well containing acetic acid and methanol in the ratio of 1:7 was added and incubated in room temperature for 5 minutes. The fixing solution was removed followed by addition of 1ml of 0.05% crystal violet stain to each well. The plates with crystal violet were incubated for 2 hours in room temperature. After incubation, the wells were rinsed with tap water. The plates were then allowed to dry on a paper towel for 24 hrs. The colonies formed were imaged and counted under light microscope (Olympus IX73).

2.5. Wound healing assay

Metastasis in cancer disease is characterized by migration of the cancer cells. To study the inhibitory effects of EP on PC3 migtation in in vitro, wound healing assay was used as described by Somaiah., et al.,²³. PC3 cells were cultured in 24 well plates and incubated in 37°C with 5% CO₂. After 100% confluence, a scratch was made at the center of each well with a 200μl pipette tip. The old media was removed and replaced with the drug media containing varying concentrations of EP such as 10μM and 25μM. The pictures were taken immediately at 0 hour followed by 24 hours and 48 hours (Olympus1X73). The % open area was analyzed using T-scratch software.

2.6. Cell cycle analysis by flow cytometry

Cell cycle analysis was performed to examine whether EP caused cell cycle arrest in PC3 cells. The experiment was performed by analyzing the DNA by staining the cells with propidium iodide as per the protocol described previously^24,25. Approximately 1×10⁴ cells in 2 ml complete media were plated per well in 6 well plate. Cells were incubated in 37°C with 5% CO₂ until 70% confluence was attained. The media was replaced with drug media containing varying concentrations of EP such as 10μM, 25μM and 50μM. After a period of 48 hours, the media was collected, the attached cells were treated with 250μl of trypsin for 5 minutes. 750μl of plain media was added and the cells were collected along with the previous collected media. The collected cells were centrifuged at 1500 rpm for 10 minutes. Supernatant was discarded and re-suspended in 70% ice cold ethanol. Samples were incubated at 4°C overnight. After incubation, cells were centrifuged at 1500 rpm for 10 minutes. Supernatant was discarded and 100μl of 50μg/ml RNAase diluted in 1X PBS was added to the cells. Samples were incubated in room temperature for 15 minutes. 100μl of 50μg/ml propidium iodide (BD Bioscience) diluted in 1X PBS was added and the samples were incubated for 30 minutes in dark. Cells were re-suspended in 300μl of 1X PBS and analyzed in a flow cytometer (FACS Calibur; Becton Dickinson, Mountain View, CA).

2.7. Real time PCR

The expression levels of various genes in different PCa cells were analyzed using real time PCR. mRNA was isolated from the cells, converted to cDNA by using high capacity reverse transcription kit obtained from Applied Biosystems. Real time PCR was carried out using SYBR green chemistry (Thermofisher scientific) as per manufacturer’s protocol. PCa cell lines were cultured in two T25 flasks each. After 90% confluency, one flask was treated with 25μM of EP and the other flask was used as control. After a period of 48 hours, the media was removed and 1ml of trizol reagent was added. After vigorous shaking, the cells were incubated in room temperature for 5 minutes and the cells were collected and transferred to a 1.5ml eppendorf tube. 200μl of chloroform was added and after shaking vigorously for 15 seconds, the solution was incubated for 2 to 3 minutes. Following incubation, the trizol/chloroform mixture was centrifuged at 14000 rpm for 15 minutes at 4°C. The upper aqueous phase which contains the RNA was transferred to a new 1.5 ml eppendorf tube. To this aqueous phase, 500μl of 100% isopropanol was added and incubated in room temperature for 10 minutes followed by centrifugation at 12000 rpm for 10 minutes. Supernatant was discarded and the pellet was washed with 1 ml of 75% ethanol by centrifuging at 10,000 rpm for 5 minutes. Ethanol was removed and the tube was air dried for 2 to 3 minutes. Pellets were re-suspended with 30μl of RNAase free water and incubated in water bath at 55–60°C for 15 minutes. cDNA was constructed from mRNA using reverse transcriptase PCR and stored at −20°C until further use. This cDNA was used for real time PCR using SYBR green assay and the expression of various stem cell markers was studied with respective gene specific primers. Relative gene expression levels were determined by calculating the fold change of treated samples (2^−ΔΔCt) comparing to control.

2.8. Alkaline Phosphatase assay

EP effects on Alkaline phosphatase (ALP) inhibition were investigated in metastatic PC3 cells using the alkaline phosphatase assay kit obtained from STEMGENT (Alkaline phosphatase staining kit II). The assay was performed as per manufacturer’s protocol with minor modifications. PCa cells were cultured in 6 well plates and were allowed to grow in 37°C with 5% CO₂ until 70% confluence. The media was replaced with drug media containing varying concentrations of EP such as 10μM, 25μM and 50μM. After a period of 48 hours, the media was removed and 2ml of PBST was added to each well and the cells were washed. 1ml of fixative solution was added to each well and incubated in room temperature for 2 to 5 minutes. Following incubation, fixative solution was removed and the cells were washed again by adding 2ml of 1X PBST to each well. After washing, 1.5 ml of the freshly prepared alkaline phosphatase staining solution was added to each well and incubated in dark at room temperature for 5 to 15 minutes. Following incubation, staining solution was removed and the cells were washed again with 1X PBS. Cells were covered with 1X PBS and observed under light microscope and the pictures were taken (Olympus1X73). The intensity of the stain was analyzed using image J software.

2.9. Apoptosis Assay

To study whether EP induces apoptosis in PC3 cells, apoptosis assay was performed. In brief, PC3 cells were stained with annexinV-FITC/PI (BD Bioscience) to examine, if the cells undergo apoptosis mediated cell death. Approximately 10,000 cells in 250μl of media were seeded per well in 8 chamber plates and incubated in 37°C with 5% CO₂ until they became 70% confluent. The media was replaced with drug media containing varying concentrations of EP (10μM, 25μM and 50μM). After a period of 48 hours, the cells were washed with 250μl of 1X PBS for 5 minutes. 2.5μl of AnnexinV-FITC and propidium iodide each were added to all the wells and incubated in dark for about 15 minutes. Cells were washed with 250μl of 1X PBS for 5 minutes. Slide was then layered with flourogel and covered with a cover slip. Cells were observed under confocal microscope and pictures were taken (Olympus 1X73). Apoptosis was also quantified in PC3 cells treated with EP (10μM, 25μM and 50μM) using flow cytometry (FACS Calibur; Becton Dickinson, Mountain View, CA).

2.10. Immunofluorescence

To detect the expression of β-catenin and cleaved caspase 3, PC3 cells were treated with EP, immunofluorescence was performed using confocal analysis. Following treatment, PC3 cells were probed with β-catenin and cleaved caspase-3 antibodies as per Velavan et al²⁶. Approximately 10,000 PC3 cells in 250μl of complete media with 10% FBS and antibiotics per well were seeded in an 8-chamber plate. After the cells reached 70% confluence, they were treated with EP of various concentrations such as 10μM, 25μM and 50μM. After 48 hours of treatment period, the media was removed and 250μl of 4% paraformaldehyde was added to each well for fixing the cells and incubated for 20 minutes. After fixing, wells were washed with 250μl of 1X PBS. Following PBS wash, 250μl of 0.1% TritonX-100 was added to the wells and incubated for 10 minutes at room temperature then washed with 1X PBS. Subsequently, 250μl of 5% BSA in PBS was added to each well and the cells were incubated for 1 hour. The cells were again washed with 250μl of 1X PBS per well followed by probing with primary antibody obtained from Cell Signaling Technology (β-catenin and cleaved caspase 3–1:200 dilution) incubated for overnight at 4°C. Following incubation, the primary antibody solution was removed and washed with 250μl of 1X PBS. After washing, 250μl of FITC-labelled respective secondary antibody (CST) 1:500 diluted in 1 % BSA in PBS was added to each well. This was incubated for an hour at room temperature. Then the secondary antibody solution was removed and 250μl of 1X PBS was added to each well followed by removal of the PBS solution. Then the slide was layered with fluorogel with DAPI (Electron Microscopy Sciences) and covered with a cover slip. The slide was analyzed and the pictures were taken using confocal microscope (Olympus FluoView FV10i). The images were quantified using image J.

2.11. Detection of intracellular reactive oxygen species (ROS) by DCFDA probe

ROS is produced by the cells undergoing oxidative stress. To determine whether EP induced production of ROS, the assay was carried out according to Gheewala., et al²⁷. Approximately 10,000 cells were seeded per well in 250μl of complete media and antibiotics in an 8 well chamber slide. After the cells attained a confluence of 70%, they were treated with varying concentrations of EP such as 10μM, 25μM and 50μM and incubated for 48 hours. After this treatment period, 10μM of DCFDA stain (sigma) was added to each well and incubated in dark for 15 minutes. After incubation, the cells were washed with 1X PBS and pictures were taken under fluorescent microscope (Olympus 1X73).

2.12. Western blotting

Western blot analysis was done as described previously^28–30. PC3 cells were seeded in T25 flasks and the flasks were incubated in 37°C with 5% CO₂ until they became 70% confluent. After 70% confluence, the flasks were treated with different concentrations of EP such as 10μM, 25μM and 50μM. After a period of 48 hours, the media in the flask was collected and centrifuged at 1500 rpm for 10 minutes. Supernatant from this centrifugation was collected, stored in −20°C and used for further analysis. The small pellet remaining was kept aside for few minutes. To the T25 flasks 5ml of plain media was added and the adherent cells were scraped with a scraper. These cells were collected in the same corresponding tubes with small pellet in the previous step. They were again centrifuged at 1500 rpm for 10 minutes. Supernatant was discarded and to the pellet remaining, 200μl of lysis buffer was added. Samples were incubated in ice for 5 minutes. They were then centrifuged at 1500 rpm for 10 minutes. 20μl of protease inhibitor was added to each sample. These lysates could be stored in −20°C for further experiments. Equal volumes of cell lysate and 2XSSB buffer were mixed together, boiled for 10 minutes at 95°C, loaded on to 10–12% SDS PAGE and the gel prepared according to the recipe and was run at 120 volts. The proteins were transferred to a nitrocellulose membrane using semi-dry transfer apparatus (Biorad) for 60 minutes at 120mAh. Presence of proteins in the membrane was confirmed using ponceau staining. Membrane was washed twice with 1X TBS and blocked using 5% skimmed milk for 1 hour. Protein expression was detected by probing with respective primary antibodies (CST) incubated for overnight at 4°C followed by secondary antibody (CST) incubated for one hour. Signals were detected using ECL (Enhanced Chemi-Luminescent) solution and developed on X-Ray films. The relative intensity of the bands comparing to control was calculated using image J software with β-actin as house-keeping gene (Sigma).

2.13. Statistical analysis

Student t-test was used for statistical analysis to find out the significance between control and treatment groups for all the experiments. P value less than 0.05 was considered significant. Graph pad prism was used for statistical analysis.

3. Results

3.1. EP differentially inhibited the proliferation of PC3 cells in a dose dependent manner

Cell viability assay is a widely used method to determine the inhibitory effects of different compounds on cancers cells. In our study, the cell viability was reduced to less than 50% in PC3 cells at a concentration of 25μM of EP. This assay suggested that EP inhibited the proliferation of PC3 cells in a dose-dependent manner (Fig 1A). The IC₅₀ value of EP for PC3 cells was found to be 25μM. In addition to the MTT cell viability assay performed in PC3 cells, we also wanted to determine if the effects of EP are blocked by inhibitors of caspase-9, caspase-3 and necrostatin (necrosis inhibitor). PC3 cells were treated with EP along with the inhibitors and the cell viability was calculated. The effects of EP were found to be significantly inhibited by caspase-9 (C9I) and caspase-3 (C3I) specific inhibitors and also necrostatin (Nec) partially rescued the effect of EP (Fig 1B), suggesting that necrosis and apoptosis may have a partial role in EP’s mechanism of action targeting PC3 cells.

Fig 1: EP treatment robustly inhibits the cell viability of PC3 cells.

A) Structure of EP. B) EP inhibited the viability of PC3 cells in a dose and time dependent manner. PC3 cells were treated with different concentrations of EP for a period of 24–72 hours and cell viability was assessed. The % cell viability was calculated and the quantitative data was shown. Dose dependent inhibition in cell proliferation was observed. Significant differences were found between control and treatment groups. C) The effects of EP were blocked by caspase-9, caspase-3 specific inhibitors and necrosis inhibitor. In PC3 cells, both the lower and higher concentration of EP (10μM and 25μM) in combination with the three inhibitors showed significant blocking effects at 48 h. Data are shown as mean ± SD (n=3). *p<0.05 compared to control group. Significant differences were found between control and treatment groups.

3.2. EP inhibited the colony formation in PC3 cells in vitro

Cancer cells have the ability to grow in colonies. The effects of EP on colony forming property of PC3 cells was tested using colony formation assay. There was a significant reduction in both the number and the size of the colonies in the EP treated PC3 cells when compared to control cells (Fig 2A). In PC3 cells, there was a significant reduction in the number of colonies starting from 10μM concentration. This assay suggested that EP was able to effectively inhibit the colony formation in PCa cells in vitro. Number of colonies were counted and plotted in graph (Fig 2B).

Fig 2: EP suppresses the clonogenic potential of PC3 cells in vitro.

PC3 cells were treated with various concentrations of EP and incubated for a period of two weeks, the developed colonies were processed by crystal violet staining followed by phase contrast imaging (Olympus 1X73). A) PC3 colonies were observed under light microscope and images were captured using digital camera. B) Quantitative representation of PC3 colonies that were plotted after counting the colonies under the microscope. Pictures were taken at 10X magnification. Data are shown as mean ± SD (n=3) *p<0.01 compared to control group. Significant differences were found between control and treatment groups.

3.3. EP exhibited anti-metastatic property in vitro, inhibited cell migration in PC3 cells

Our data showed that EP inhibit the viability and colony formation of PC3 cells. Next, we wanted to determine if EP could inhibit the migratory properties of PC3 cells. Wound healing assay demonstrates the process of metastasis in vitro. The % open area calculated using the T-scratch software showed that there was a significant difference between the control and the treated wells. At 0 hour, all the wells were found to have approximately the same open area wound made by the pipette tip. As there was an increase in time from 0 hour to 24 and 48 hours, the closing of the wound became slower in the treated cells, whereas control cells grew and closed the wound much faster (Fig 3A). At the end of 48 hours, the % open area in PC3 control was 0.54% whereas the EP 25μM treated well had an open area of 38.67%. The results from this assay suggested that EP effectively inhibited the migration of PC3 cells.

Fig 3: EP inhibited the migration property of PC3 cells in vitro.

PC3 cells were cultured and a scratch was made at the center of each well followed by treatment with EP. Following 48 hours of incubation, pictures were taken every 24 hours starting with 0 hour. Time and dose dependent effects of EP were observed. A) The % of open area in PC3 cells was found to be decreased in control as time progresses whereas EP treatment inhibited migration and the % open area did not decrease significantly. B) Quantitative graph of PC3 cells at different time points. Data are shown as mean ± SD (n=3) *p<0.01 compared to control group. Significant differences were found between control and treatment groups.

3.4. EP caused G0/G1 cell cycle arrest in PC3 cells

As we have shown that EP can inhibit cell proliferation, colony formation and migration of PC3 a cells, we wanted to study if EP could induce cell cycle arrest. PC3 cells were treated with EP, stained with propidium iodide and analyzed using flow cytometry. The results from the cell cycle analysis showed that PC3 cells were arrested in the G0/G1 phase of the cell cycle (Fig 4). The % of cells in each phase of the cell cycle was analyzed using Flow Jo software. In PC3 cells, there was a significant increase in the % of cells in the G0/G1 phase from 10μM of EP treatment onwards when compared to control.

Fig 4: EP targets PC3 cells via inducing G0/G1 phase cell cycle arrest.

The CRPC cell line, PC3 was treated with EP of different concentrations and stained with propidium iodide. Following staining, the cells were analyzed using flow cytometry A) Histograms showing the profiles of PC3 control cells and EP treated cells analyzed using Flow Jo software. B) Quantitative data of PC3 showing % of cells in each phase of cell cycle. Results shown significant cell cycle arrest in G0/G1 phase were seen from 10μM of EP treatment onwards. Data are shown as mean ± SD (n=3), *p<0.01 compared to control group. Significant differences were found between control and treatment groups.

3.5. EP inhibited the expression of cancer stem cells (CSCs) markers of PC3 cells in vitro

Cancer stem cells (CSCs) have the inherent ability to be resistant to conventional chemotherapeutic drugs³¹. Therefore, targeting these CSCs would be an effective therapy for advanced PCa. Hence, we sought to explore if EP could target stem cell properties of PCa cells. In order to accomplish this, first we performed real-time PCR to analyze the mRNA expression levels of known stem cell markers. Initially, the expression profiles of various cancer stem cell markers like Nanog, Oct3/4, Sox-2 and ALDH1 were screened in different PCa cell lines such as PC3, DU145, LNCaP, VCaP, C4–2, and 22RV1. It was observed that Nanog was over-expressed in LNCaP, VCaP, 22RV1 and DU145 (Fig 5A) whereas Oct3/4 was found to be over-expressed in VCaP and DU145 cells (Fig 5A). Similarly, Sox-2 was significantly over-expressed in PC3 and DU145 cell lines (Fig 5A). PCa cell lines such as C4–2, 22RV1, PC3 and DU145 also showed over-expression of ALDH1 (Fig 5A). RWPE-1, which is a non-cancerous prostate cell line was used as a control to compare the expression profiles of different cancer stem cell markers in PCa cell lines. Next, we desired to examine if EP could inhibit the expression of these cancer stem cell markers in PC3 cell line. From the results, it was seen that EP inhibited the expression of the CSCs markers Nanog, Oct3/4, Sox-2 and ALDH1 significantly (Fig 5B). Furthermore, we studied the expression level of β-catenin, CD44, cyclin-D1 and c-Myc expression in EP treated PC3 cells, which have various functions in cell division, migration, and stemness. Upon treatment with EP, expression level of β-catenin, CD44, cyclin-D1 and c-Myc were downregulated in PC3 cells (Fig 5C). The concentration of EP used for these experiments was 25μM. β-Actin was used as an internal control.

Fig 5: Stem cell markers are differentially expressed in various prostate cancer cell lines.

A) Quantitative expression profile of various cancer stem cell markers in different PCa cell lines. The mRNA from PCa cell lines such as LNCaP, C4–2, 22RV1, VCaP, PC3 and DU145 and the non-cancerous prostate cell line RWPE-1 was collected, followed by conversion of mRNA to cDNA using reverse transcription PCR. The expression of the different cancer stem cell markers was analyzed using real time PCR. Our results indicated that Nanog was found to be overexpressed in 22RV1, VCaP, DU145 and LNCaP. Oct3/4 was found to be overexpressed significantly in DU145 and VCaP cells. Similarly, Sox-2 was overexpressed in significant PC3 cell lines. ALDH1 was overexpressed several folds in 22RV1, C4–2, PC3 and DU145. RWPE-1 was used as a control to calculate the relative expression levels of the different CSC markers. Relative quantification of the expression of the CSC markers in comparison to control was performed by calculating the fold change (2^−ΔΔCt). B) EP diminished the expression of various cancer stem cell markers in PC3 cells. EP significantly reduced the expression of CSC markers such as Nanog, ALDH1, Oct3/4, and Sox-2. C) EP inhibits the expression of various genes involved in regulation of cancer stem cell properties. The expression levels of β-catenin and CD44, cyclin D1 and c-Myc were significantly reduced by EP treatment in PC3 cell line. Data are shown as mean ± SD (n=3). *p<0.05 compared to control group. Significant differences were found between control and treatment groups.

3.6. EP inhibited alkaline phosphatase expression in PC3 cells in vitro

Alkaline phosphatase is a hydrolase enzyme which would dephosphorylate molecules such as nucleotides, proteins and alkaloids under alkaline conditions and it is highly expressed in CSCs³². Therefore, alkaline phosphatase assay has been widely used for detecting undifferentiated pluripotent stem cells³³. When PC3 cells were treated with EP of different concentrations, it was found that EP effectively inhibited the expression of alkaline phosphatase enzyme activity (Fig 6). Significant decrease in the staining intensity of alkaline phosphatase enzyme activity was observed in 25μM and 50μM concentration of EP in PC3 cells. This data suggested that EP could target the CSC properties in vitro.

Fig 6: EP effectively reduced the activity of alkaline phosphatase enzyme.

PC3 cells were treated with EP of different concentrations and stained for detecting the expression of alkaline phosphatase enzyme. A) The staining was found to be reduced in the treated cells when compared to control in PC3 cells. Pictures were taken under 20X magnification. B) The intensity of alkaline phosphatase stained cells were quantified using ImageJ software. Data are shown as mean ± SD (n=3). *p<0.05 compared to control group. Significant differences were found between control and treatment groups.

3.7. EP induced apoptosis in PC3 cells

Apoptosis plays a major role in the initiation and progression of cancer as well as in cancer therapy. Many of the chemotherapeutic, anti-cancer agents work by activating the apoptosis pathway. To detect apoptosis, the PC3 cells were treated with EP and stained with annexin V- FITC/PI to detect apoptosis. The fluorescence intensity of annexin V-FITC correlates to the cells undergoing apoptosis. The results from this study revealed that the intensity of the green fluorescence increased as the concentration of EP increased (Fig 7A and 7B). This observation was further confirmed by flow cytometry data (Fig 7C). The significant increase in the relative annexin V-FITC binding was found to be at 25 and 50μM concentration in PC3 cells (Fig 7B and 7D). These results confirm that the PC3 cells were undergoing apoptosis when treated with EP.

Fig 7: EP treatment induces apoptotic cell death in PC3 cells.

To study the effect of EP induced apoptosis, PC3 cells were treated with different doses of EP. Following treatment, apoptosis was detected using annexin-V FITC/PI staining. A) Dose dependent increase in the intensity of annexin-V was observed in PC3 cells after EP treatment. Pictures were taken under 20X magnification. B) Data represents the ImageJ quantification showing relative annexin binding levels in PC3 cells. C) EP induced potent apoptosis in a dose dependent manner as determined by flow cytometry analysis. D) Representative graph of flow cytometry results showing percent apoptosis in PC3 cells treated with various doses of EP. Data are shown as mean ± SD (n=3) *p<0.01 compared to control group. Significant differences were found between control and treatment groups.

3.8. EP modulated the cellular localization of β-catenin in PC3 cells

The Wnt/β-catenin signaling pathway plays a key role in the chemo-resistance of various cancers^34,35. In case of non-cancerous cells, the β-catenin is located in the cytoplasm in an unstable form and it is destroyed by the process of ubiquitination. However, in the cancer cells, β-catenin becomes stabilized and translocates into the nucleus where it binds to the Tcf and regulates the expression of various downstream target genes³⁴. PC3 were treated with EP and analyzed for the expression and location of β-catenin. The images of EP treated cells analyzed using confocal microscope revealed that there was translocation of β-catenin from the nucleus to the cytoplasm (Fig 8A). Intensity of β-catenin staining was quantified using ImageJ analysis (Fig 8B).

Fig 8: EP treatment impedes β-catenin nuclear expression in PC3 cells.

EP treatment caused translocation of β-catenin. After treating PC3 cells with EP for 48 hours, the cells were stained with β-catenin antibody conjugated with FITC. A) The intensity of green florescence decreased specifically in the nucleus with EP treatment in PC3 cells. DAPI was used as a counter stain to detect the nucleus. Pictures were taken under confocal microscope (Olympus FluoView FV10i) 60X magnification. B) Image shows quantitative expression of β-catenin in PC3 cell line. Data are shown as mean ± SD (n=3). *p<0.05 compared to control group. Significant differences were found between control and treatment groups.

3.9. EP promoted caspase-3 cleavage in PC3 cells

Caspase-3 is one of the critical and reliable makers for apoptosis. Since caspase-3 inhibitor was previously shown to block the effects of EP, we wanted to detect the expression of cleaved caspase-3 (active form) in PC3 cells treated with EP. From the results, it was inferred that the expression of cleaved caspase-3 increased as the concentration of EP increased (Fig 9). The significant increase in the expression of cleaved caspase-3 was observed at 25μM and 50μM concentration of EP in PC3 cells. Confocal microscope was used to analyze the pictures and relative expression of cleaved caspase-3 was calculated using image J software (Fig 9B).

Fig 9: EP treatment promoted caspase-3 activation in PC3 cells at the cellular level.

PC3 cells was treated with EP of various doses and incubated for 48 hours. After incubation, the cells were probed for cleaved caspase-3 antibody conjugated with FITC. A) PC3 cells showed increased cleaved caspase-3 expression in treated cells when compared to control cells. Pictures were taken under confocal microscope (Olympus FluoView FV10i) 60X magnification. B) ImageJ quantification data showing the relative expression of cleaved caspase-3 comparing to control. Data are shown as mean ± SD (n=3). *p<0.01 compared to control group. Significant differences were found between control and treatment groups.

3.10. EP induced ROS production in PC3 cells

ROS is generated and released by the cells which undergo oxidative stress³⁶. The conventional chemotherapeutic PCa drugs Docetaxel and Cabazitaxel have been shown to induce ROS formation in CRPC cells³⁶. To study if EP could induce the formation of ROS, PC3 cells were treated with EP of different concentrations, stained with DCFDA (ROS stain) and analyzed using fluorescent microscope. In PC3 cells, 25μM and 50μM concentration of EP showed increased fluorescence when compared to control indicating that EP induced ROS generation (Fig 10A). The relative ROS levels were calculated using ImageJ software (Fig 10B).

Fig 10: EP induced oxidative stress in PC3 cells via generation of ROS.

PC3 cells were treated with different doses of EP and stained with DCFDA. A) PC3 cells showed increased fluorescence in EP treated samples when compared to control. Pictures were taken under microscope (Olympus 1X73) 10X magnification. B) Quantitative data showing relative ROS levels in PC3 cells. Significant increase was observed at 25μM and 50μM concentration. The significant increase in intensity was found starting from 25μM concentration of EP. Data are shown as mean ± SD (n=3). *p<0.01 compared to control group. Significant differences were found between control and treatment groups.

3.11. EP downregulated the expression of various cell cycle markers in CRPC cell lines, key anti-apoptotic markers, upregulated the expression of DNA damage marker, apoptotic markers and activates caspase-9

Based on our previous experiments, it is evident that EP shows anti-cancer effects against PC3 cells. To further analyze the mechanism of action of EP targeting PC3 a cells, Western blotting analysis was performed to detect the expression levels of different cell cycle markers such as cyclin D1, cyclin D3, CDK4 and c-Myc. The results showed that EP effectively downregulated the expression of cyclin D1, cyclin D3, CDK4 and c-Myc (Fig 11A).

Fig 11: EP diminished the expression of cell cycle markers, anti-apoptotic markers and induced caspase dependent apoptosis in PC3 cells.

PC3 cells were treated with EP of different doses for a period of 48 hours and subjected to western blotting analysis. A) The bands showing intensity of cyclin D1, cyclin D3, CDK 4 and c-Myc in PC3 treated with EP. B) Quantification of the data using image J software. Dose dependent decrease in the intensity of the bands were observed for the markers in PC3 cells. EP treatment decreased the expression of IAP family proteins with complete inhibition at 50μM concentration of EP demonstrating that EP effectively reduced the expression of anti-apoptotic proteins. C) The expression levels of Mcl-1, survivin, XIAP, and c-IAP1 were downregulated by EP treatment in PC3 cells. D) Relative expression levels of the anti-apoptotic markers in EP treated PC3 cells when compares to control. E) The bands showing pro-apoptotic caspase protein intensity of each marker in PC3 cell line. Clear activation of caspase-9 was observed along with activation of Bad, pH2A.X, PARP-1 and cleaved PARP-1. These results suggest that EP targeted PCa cells by a mechanism of caspase mediated cell death. F) Relative expression levels of various markers in PC3 cells treated with EP comparing to control.

Western blotting analysis was carried out to study the expression of IAP family of proteins like XIAP, c-IAP2 and survivin in PC3 cells treated with EP. The results from this experiment demonstrated that EP reduced the expression of survivin, XIAP and c-IAP2 (Fig 11C). EP treatment was able to effectively diminish the expression of Mcl-1 while having modest impact on the expression of TCTP in PC3 cells. Since our cell viability results using caspase-9 and 3 specific inhibitors, showed an indication that caspase-9 and 3 play a role in the mechanism of EP’s action, we studied the expression of various caspases by western blotting. EP treatment seems to activate caspase-9 while also increasing the activation of other caspases such as caspase-8, caspase-7 and caspase-3 by downregulating their pro-caspase levels (Fig 11E). The results suggested that EP upregulated the expression of pH2A.X and a pro-apoptotic marker, Bad was increased several folds comparing to control in PC3 cells (Fig 11E). The cleavage of PARP-1, an important indicator of apoptosis marker was found to be increased in the EP treated PC3 cells (Fig 11E).

4. Discussion

PCa is one of the most common cancer among men in United States¹. Current treatment options available for PCa include surgery, radiation therapy, chemotherapy and hormonal therapy¹. However, patients develop resistance and show toxicity with the current available treatments^1–3. Chemotherapeutic drugs administered for PCa like Docetaxel, Cabazitaxel, and Abiraterone have adverse side effects on patients^2,4. Hence, there is an urgent need for developing new treatment strategies against PCa. The results from cell viability assay demonstrated that EP effectively inhibited the cell viability of PC3 cells (Fig 1). A previous study using other derivatives of Avermectin such as Ivermectin, Doramectin and Selamectin has shown the inhibition of cell viability in human colon cancer cell lines³⁷. Similarly, Ivermectin and Selamectin has been shown in a study to effectively reduce the cell proliferation of triple negative breast cancer cell line¹⁸. In our study, EP which is also a derivative of avermectin shows significant inhibition of cell proliferation in PC3 cells. After its ability to effectively inhibit the proliferation of PC3 cells, the clonogenic potential and migratory potential of the PC3 cells in the presence of EP treatment was studied. The results from this experiment suggested that EP effectively inhibited the colony forming property of PC3 cells (Fig 2). EP also exhibited anti-metastatic property inhibiting the migration of PC3 cells significantly (Fig 3). In previous studies, Ivermectin and Selamectin has been shown to inhibit the clonogenic and self-renewal properties of colon cancer cells and breast cancer cells^18,37. Thus, EP has the potential as an anti-cancer agent for targeting metastatic PC3. Analysis of β-catenin expression by confocal microscopy suggested that there was translocation of β-catenin from the nucleus to the cytoplasm in EP treated PC3 cells indicating the involvement of β-catenin signaling pathway (Fig 8). A previous study on colon cancer indicated that the drug Ivermectin which is approved for human use, showed anti-cancer activity by blocking wnt/β-catenin pathway³⁷.

Cancer stem cells (CSCs) constitute a malignant sub population in tumors. They have the inherent ability to self-renew and their role in cancers is associated with drug resistance and cancer relapse³⁸. There are several recent reports suggesting that PCa may originate from CSCs^39,40. A recent study on colon cancer reports the role of canonical Wnt/β-catenin signaling pathway showing reduced expression of stem cell markers ASCL2 and LGR5 with Ivermectin treatment³⁷. Embryonic stem cells within the cancer tissue expressing markers such as ALDH1, Oct3/4, Nanog and Sox-2 are considered to have clonogenic, proliferative and tumor initiating potential³⁸. Identification and targeting of these cells would be a potent way for treating metastatic cancers. CSCs are known to have elevated levels of an intracellular enzyme, alkaline phosphatase. Therefore, in the present study, the presence of undifferentiated CSCs in PC3 cells was detected by alkaline phosphatase assay. EP treatment inhibited the activity of alkaline phosphatase enzyme (Fig 6). Our results demonstrated that EP treatment caused significant reduction in the mRNA expression levels of several cancer stem cell markers such as ALDH1, Nanog, Oct3/4 and Sox-2 in in PC3 cells as compared to control RWPE-1 cells (Fig 5B). These results suggested that EP has the potential to target CSCs and could be an effective anti-cancer agent against advanced PCa cells.

Reactive Oxygen Species (ROS) are radicals or ions which have one free electron in their last shell and this makes them highly reactive. An increase in the intracellular ROS can induce cell cycle arrest, apoptosis or necrosis⁴¹. ROS is essential for various biological processes in normal cells. On the contrary, in cancer cells generation of ROS causes cell death. Due to this dichotomous role of ROS, both pro and anti-oxidant therapies have been proposed for cancers⁴². A very recent study which evaluated the efficacy of Ivermectin on leukemia cells showed that Ivermectin increased the intracellular levels of ROS and caused apoptosis in leukemia cells⁴³. Results from our study showed that there is a significant increase in ROS production at 25μM and 50μM of EP treatment in PC3 cells (Fig 10). EP induced production of ROS in PC3 cells was found to be accompanied with G0/G1 cell cycle arrest and eventual cell death. Apoptosis is a form of programmed cell death which is deregulated in cancer cells. Results from annexin-V/PI staining demonstrated that PC3 cells undergo apoptosis with increasing treatment doses of EP (Fig 7). A similar study evaluating apoptosis induced by Emamectin benzoate in K562 and Molt-4 leukemia cells showed that the number of apoptotic cells increased with treatment when compared to control cells⁴⁴. Hence, the results from our study supports the notion that EP induced ROS formation, G0/G1 cell cycle arrest and apoptosis in PCa cells.

Western blotting analysis revealed the further underlying mechanisms involved with EP targeting PCa cells. EP resulted in downregulation of various cell cycle markers such as cyclin D1, cyclin D3, CDK4 and c-Myc (Fig 11A). These proteins are involved in the regulation of G1 phase of the cell cycle⁴⁵. This finding was in correlation with the results from cell cycle analysis where the EP treatment caused G0/G1 cell cycle arrest in PC3 cells (Fig 4). Our observation that EP inhibiting the nuclear localization of β-catenin suggests that its downstream target genes such as cyclin D1 and c-Myc could have been down regulated by EP via counteracting β-catenin signaling. Further studies are needed to confirm these findings. Various anti-cancer drugs target cancers by inhibiting the activity of the anti-apoptotic proteins like survivin which belongs to Inhibitor of Apoptosis (IAP) family. The overexpression of survivin in tumor cells has been positively correlated with the development of multidrug resistance. Survivin is therefore considered to be a cancer biomarker and an attractive therapeutic target⁴⁶. The most widely studied survivin inhibitor, YM155 which was initially discovered by Astellas Pharma inhibits survivin expression by diminishing survivin promotor activity. YM155 is currently in phase II clinical trials⁴⁷. There are several other survivin inhibitors such as UC-112 and derivatives, fl118 and shepherdin which also shows anti-tumor activity in various cancer cell lines and xenograft models. However, clinical study of these compounds on patients is not reported⁴⁸. Moreover, Embelin, a natural product derived from Embelia ribes plant has been identified as an inhibitor of IAP family of proteins and targets PCa⁴⁹. Another natural product namely hydroxycampothecin which is isolated from a Chinese tree, shows anti-cancer activity in colon cancer cells by downregulating the expression of IAP family of proteins⁴⁶. EP also downregulated the expression of IAP family of proteins such as survivin, XIAP and c-IAP1 in PC3 cell line (Fig 11C). EP also significantly reduced the expression of Mcl-1 which is a constituent member of Bcl-2 family of anti-apoptotic proteins (Fig 11C). Mcl-1 has been shown to be an important regulator of apoptosis in PCa cells. Mcl-1 has the ability to protect PCa cells from the cell death inducing effects of endocrine therapy⁵⁰. It has been reported previously that a combination of Genistein present in soy product and Cabazitaxel showed anti-cancer effects in CRPC cell lines with a significant inhibition in Mcl-1 protein expression⁵¹. In this study, we have also tested the impact of EP on TCTP expression which is an antiapoptotic protein and its overexpression correlates with CRPC progression⁵². Our data showed that anticancer effect of EP in PC3 cells appears to be independent of targeting TCTP. However, the pro-apoptotic marker Bad was observed to be significantly increased with EP treatment in PCa cell lines (Fig 11E). Further, EP also caused downregulation of different pro-caspases like caspase-3, 7, 9 and 8 in PC3 cells (Fig 11E) suggesting that activation of these caspases by EP. Of note, our study has demonstrated the activation of caspase-3 by EP in PC3 cells as determined by confocal analysis. In line with this finding, EP also activated caspase 9 in PC3 cells as demonstrated by cleaved caspase-9 suggesting that EP induced apoptosis in PC3 cells via caspase dependent mechanism (Fig 11E). EP treatment also resulted in the activation of DNA damage response marker, pH2A.X implicating that EP caused DNA damage as well in PC3 cells (Fig 11E). Thus, results from the western blotting experiments revealed different types of anticancer mechanisms by which EP would target PCa cells.

Conclusion

Based on our studies, it was observed that EP has anti-cancer activity against PC3 cells. According to the results from our pre-clinical study, it is evident that EP has the potential to be an effective anti-cancer therapy for advanced PCa. However, in vivo studies need to be carried out to determine the detailed anti-cancer effects of EP for potential use in clinics.