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FCX, an arylidene derivative, induces apoptosis in androgen receptor-selective prostate cancer cells

1 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia
2 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia; Department of Biochemistry, Faculty of Science, Alexandria University, Alexandria, Egypt
3 Department of Microbiology and Parasitology, Center for Stem Cell Research, College of Medicine, King Khalid University, Abha, KSA

Correspondence Address:
Rajagopalan Prasanna,
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha
Saudi Arabia
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_228_17

 > Abstract 

Context: Rational screening of arylidene derivatives for biological activities has resulted in many lead molecules with anticancer properties with effective therapeutic window.
Aims: In the current study, FCX, an arylidene derivative, was screened for anticolon and prostate cancer activity.
Settings and Design: Prostate and colon cancer cell lines were used to check the FCX effect on proliferation, apoptosis, and mechanism of drug action.
Subjects and Methods: LNCaP, PC-3, HCT-8, and HT-29 cells were treated with various concentrations of the FCX. MTT assay was performed to check proliferation, propidium iodide and Hoechst dual staining for DNA fragmentation, and Annexin V binding assay for apoptosis, and cell cycle assay was done using flow cytometry. Functional androgen-mutated receptor cells were used mechanistic pathway elucidation.
Statistical Analysis Used: A minimum of three individual replicates at different time periods were taken as mean value. The data were expressed in mean ± standard deviation. Student's t-test and one-way ANOVA were used to assess the statistical difference between the groups.
Results: FCX inhibited proliferation of prostate cancer cell lines in a dose-dependent manner with more selectivity toward LNCaP cells. Nuclear fragmentation and dose-dependent increase in Annexin V-positive LNCaP cells revealed apoptosis. Cell cycle G2/M phase arrest along with sub-G0/G1 population augmented the antiproliferative observations. Addition of FCX in the presence of estradiol, testosterone, and dehydroepiandrosterone, LNCaP cells markedly caused a dose-dependent increase in cell proliferation indicating the compound activity to be facilitated through androgen receptor pathway.
Conclusions: Together with the results, it is evident that FCX has a wide therapeutic window in the in vitro inhibition of the prostate cancer cells mediated by hormone-dependent effects.

Keywords: Apoptosis, drug design, cell cycle arrest, FCX, LNCaP, prostate cancer

How to cite this URL:
Prasanna R, Elbessoumy AA, Chandramoorthy HC, Dera A, Al Fayi M. FCX, an arylidene derivative, induces apoptosis in androgen receptor-selective prostate cancer cells. J Can Res Ther [Epub ahead of print] [cited 2019 Nov 21]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=269051

 > Introduction Top

Prostate cancer is the second major cause of cancer-related mortality in men worldwide.[1] Current treatment options are restricted to traditional chemotherapy, surgery, radiation, or hormone therapy. Although the survival rates are roughly about 5 years postdiagnosis in 95% cases, the selection of the therapeutic option is often fraught with several side effects,[2] and metastasis of the prostate cancer considerably reduces the survival rate. Advanced prostate cancer, therefore, often becomes complicated to treat with side effects of the chemo-radiotherapy, worsening the overall quality of life. The mechanism behind the resistance of the prostate cancer is the cancer stem cells in the niche, which becomes dormant, while the chemotherapy targets active replicating cells.[3] The progressive late stage of prostate cancer becomes androgen independent on the whole and may not respond to hormone ablation therapy, thus making poor prognosis. Selection of the cell lines to study prostate cancer or screen suitable drugs is ambiguous that prostate cancer is expansive and arises from several cell sources in the prostate itself. LNCaP, PC3, and DU-145 cells are some of the well-characterized prostate cancer lines available, and our studies were conducted using androgen-dependent LNCaP and androgen-independent PC3 cell lines. Recently, there is a huge transition for selection of the drug molecules targeting key metabolic pathways, which has increased the scope of efficient and safe treatment of prostate cancer. Further, the targeted therapies have contributed more to identify the key factors promoting tumorigenesis and attenuate the pathway either inducing cell death or skewing the cells to senescence. Although there are many reports on a number of small molecules inhibiting the critical cancer targets, there is always a need to develop safe and economically viable compounds as great value addition in the current existing high-cost prostate cancer treatment regimen. Few previous studies have shown small molecules inhibiting prostate cell growth by inducing apoptosis and cell cycle alterations leading to cell death. However, these studies did not indicate in elaborate on the fate of the androgen-dependent prostate cancer cells and androgen-independent prostate cell lines.[4],[5]

Therefore, the selection of the small molecules as a drug for prostate cancer should be active and classified based on hormone dependence. Arylidene indanones belong to this class of economical and safe compounds with potential anticancer properties in various types of cancers investigated by us and others. Further Indanones are shown to reverse the multidrug resistance in cells caused by standard anticancer drugs and synergistically acted with the drugs to stop cancerous cell proliferation.[6] It is previously reported 2-arylidene-4, 7-dimethyl indan-1-one (FXY-1) to be effective against breast adenocarcinoma cells by G2/M cell cycle arrest and further inducing DNA damage in the lung cancer models leading to extensive apoptosis through the mitochondrial pathway.[7],[8] Based on the previous observations, we synthesized structural analogs by rational drug design to arrive at a few promising lead molecules which were identified with preliminary screening procedures. Herein, the in vitro activity of a novel, synthetic, small molecule FCX of arylidene indanones class is reported with demonstrable significant anticancer activity on prostate cancer cells. The synthesis method of FCX is indicated in [Figure 1]. Briefly, arylidene moiety was first prepared and then the indan-1-one framework was constructed.
Figure 1: Chemical structure and synthesis of FCX

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 > Subjects and Methods Top

All chemicals and reagents were purchased from Sigma (St. Louis, MO) unless otherwise indicated. LNCaP, PC3, HCT-8, and HT-29 cell lines were obtained from the American Type Culture Collection. Annexin V kit was purchased from eBioscience, USA. Guava cell cycle reagent was obtained from Millipore Corp, USA.

Cell culture

All cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 U/ml streptomycin. Cells (passages 3–12) were maintained in a humidified atmosphere of 5% CO2 incubator at 37°C until the confluence stage was attained. The medium was replaced every 2 days, and the maintenance was strictly in accordance with the standard methods. Experiments commenced after 70% confluence was attained.

Cell Proliferation assay

Proliferation assay was performed as described by Mosmann[9] with modifications. About 5000 cells/well in 100 μl of RPMI media supplemented with 10% FBS and 1% pen-strep were plated in triplicates in a 96-well plate. Cells were incubated overnight, and 50 μl of the compound containing the desired final concentration was added along with a DMSO blank. The cells were incubated at 37°C and 5% CO2 for 72 h. About 15 μl of 5 mg/ml MTT was added and incubated for 3.5 h. Media was aspirated and MTT was dissolved in 150 μl of DMSO, and absorbance was read at 560 nm with reference at 640 nm. Percent inhibition was calculated after subtracting day 0 MTT read. Results were analyzed using GraphPad Prism software (La Jolla, CA, USA).

Morphological analysis (dual staining)

Propidium iodide (PI)/Hoechst 333258 dual staining was done as described by Belloc et al. 1994[10] with a small variation. LNCaP cells were grown on coverslips placed in Petri dishes, at a concentration of 1 million cells per plate. Sterilized Petri plate and coverslips were used. The plates were incubated for a period of 48 h to obtain a sheet of cells and then treated with respective concentration of FCX. After 24 h, the cells were washed with cold phosphate-buffered saline, and cell suspension was made with the media. Two microliters of combined dye of 100 mg/ml PI and 100 mg/ml Hoechst 333258 was added to 20 ml of cell suspension, and 5 ml of stained solution was transferred to a glass slide for immediate analysis using a fluorescence microscope.

Annexin V assay

The assay was performed using Annexin V detection kit from eBiosciences, USA, as per the manufacturer's instructions as follows. 0.5 × 106 cells were grown in 6-well plate with complete growth media for 24 h until they are semi-confluent. Cells were treated with respective concentrations of FCX and incubated in CO2 incubator for 72 h. Harvested cells were washed twice with wash buffer and incubated with 0.25 μg/ml Annexin V reagent in 1X binding buffer for 15 min. After a couple of washes with wash buffer, cells were re-suspended back in binding buffer containing 0.5 μg/ml PI and 10,000 events acquired on a Guava easyCyte™ flow cytometer. Data were analyzed using InCyte software, Millipore. Early- and late-phase apoptotic cells were segregated with a quadri-plot graph, and total percentage of apoptotic cells was represented using GraphPad Prism software (La Jolla, CA, USA).

Cell cycle analysis

LNCaP cells were seeded at a density of 0.5 × 106 cells per well in a 6-well plate and incubated with FCX at desired concentrations for 72 h at 37°C in a 5% CO2 incubator. After incubation, cells were fixed in 70% ethanol and stored at −20°C until analysis. Cells were stained with Guava® cell cycle reagent according to the manufacturer's instructions, and 10,000 events were acquired on a Guava easyCyte™ flow cytometer. Data were analyzed using Express Pro software (Millipore, USA), and percentage cell population in different cell cycle stages with respect to control was calculated.

Statistical analysis

Experiments were carried out at least in triplicate, and results are expressed as the mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 6.0 (La Jolla, CA, USA). GI50 values were calculated using a nonlinear regression fit model with variable slope and plotted accordingly. Differences between two groups were analyzed using the two-tailed Student's t-test, whereas those between three or more groups were analyzed using one-way ANOVA comparisons. Differences with P < 0.05 (*) were considered statistically significant.

 > Results Top

Antiproliferative activities and selectivity of FCX on prostate cancer cells

A library of small molecules was synthesized through rational drug design and screened for activity in prostate and colon cancer cells by analyzing antiproliferative activity by MTT assay at the end of 72 h. FCX [Figure 1] caused a dose-dependent reduction of LNCaP cells with a GI50 of 345.7 nM [Figure 2]a which was found to be greater than 5-fold potent as compared to gemcitabine in LNCaP cells [Figure 2]a. The normal-nontumorigenic colon cancer cells (CCD-18Co) were very much tolerant to FCX treatment with GI50 value of 8464 nM [Figure 2]b. We next tested the molecule in PC3 cells. The compound inhibited the growth of PC3 cells at a higher concentration when compared with LNCaP cells [Figure 2]c. In addition to this, the compound also affected the growth of colon cancer cells (HCT-8 and HT-29 cell lines) albeit at a higher concentration [Figure 2]d. As a result of higher efficacy and selectivity, LNCaP cell line was used for further studies.
Figure 2: Antiproliferative profile of FCX in prostate and colon cancer cell lines. (a) GI50 for FCX and gemcitabine in LNCaP cells. (b) GI50 of FCX in normal nontumorigenic colon cancer cells. (c) GI50 of FCX in LNCaP and PC3 cells (d) GI50 of FCX in HCT-8 and HT-29 colon cancer cells

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FCX-induced nuclear fragmentation and apoptosis in prostate cancer cells

We next assessed the nuclear changes of prostate cancer cells upon FCX treatment. After FCX treatment and subsequent staining with PI and Hoechst 333258, LNCaP cells showed apoptotic bodies characterized by cytoplasmic condensation/degradation and fragmented nuclei [Figure 3]a and [Figure 3]b. As the cell membrane after drug treatment becomes vulnerable, the dye enters the cell and stains the condensed/fragmented nuclei which are indicated in the figure. To substantiate this observation and to check if it is by the apoptotic inducing property of FCX, Annexin V binding efficacy of compound-treated cells was analyzed by flow cytometry. FCX increased early- and late-phase apoptosis in LNCaP cells [Figure 3]c. A dose-dependent increase of total apoptotic cells was also observed upon treatment at the end of 72 h [Figure 3]d.
Figure 3: Induction of apoptosis of FCX on prostate cancer cell line. (a and b) Ability of FCX to induce Nuclear fragmentation in LNCaP cells determeined by Hoechst 333258 and propidium iodide staining. Blue color represents intact nuclei and pink color are indications of fragmentation in nuclei. All experiments were performed minimum thrice unless stated and representative results are shown. (c) Flow cytometric enumeration of the apoptotic cells by Annexin V staining showing early- and late-phase apoptosis. (d) Dose-dependent increase of total apoptotic cells in LNCaP cell lines on treatment with various concentration of FCX. Results expressed as mean ± standard deviation *P ≤ 0.05

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Cell cycle changes by FCX

Next, cell cycle changes were analyzed in LNCaP cells after 72 h treatment of FCX. An increase of sub-G0/G1 cells and G2/M phase cells with respect to control [Figure 4]a was observed in the LNCaP cells. For 10,000 nM FCX treatment, almost 5-fold increase in the sub-G0/G1 cells increased was observed compared to that of control [Figure 4]b. A 4-fold increase in G2/M phase cell population was also evident at the same concentration [Figure 4]c. The effect was found to be dose dependent at lower concentrations [Figure 4]b and [Figure 4]c.
Figure 4: (a) Flow cytometric analysis of cell cycle changes after 1000, 5000, and 10,000 nM FCX treatment in LNCaP cells for 72 h. Representative histograms from several repeats of the experiment are shown. (b and c) Percentage cell population in different cell cycle stages with respect to control. Results expressed as mean ± standard deviation *P ≤ 0.05

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Androgen-responsive proliferation

The steroid-responsive human LNCaP prostate cancer cell line, containing a functional but mutated androgen receptor (AR), was used to compare effects of dehydroepiandrosterone β-estradiol (E2), testosterone (T), and dihydrotestosterone (DHT) on cell proliferation. LNCaP cells were incubated with desired concentrations of E2, T, and DHT for a period of 3 days, and induction of cell growth was determined using a MTT reduction assay. Addition of E2, T, and DHT resulted in a dose-dependent increase of LNCaP cell proliferation (data not shown). While addition of 1 nM T and DHT markedly increased cell proliferation of 38% and 55% compared to control, 10 nM E2 increased the proliferation by 31% [Figure 5]a. LNCaP cells were next incubated with desired concentrations of either compound alone and/or induced with 10 nM of estradiol, 1 nM of testosterone, and 1 nM of DHT and incubated for 72 h. Growth inhibition was determined by a MTT reduction assay. Addition of T, DHT, or E2 along with FCX to LNCaP cells resulted in a significant shift of GI50 of the compound [Figure 5]b.
Figure 5: (a) Induction of LNCaP cells by β-estradiol, testosterone, and dihydrotestosterone after 3-day treatment. Results expressed as mean ± standard deviation *P ≤ 0.05. (b) GI50 of FCX in prostate cancer cells after 10-nM β-estradiol/1-nM testosterone/1-nM dihydrotestosterone. Addition of testosterone/β-estradiol/dihydrotestosterone resulted in significant shift of GI50 indicating a protective effect over FCX by these steroids in prostate cancer cells

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 > Discussion Top

Advances in cancer research start from the search for anticancer agents which has a safer therapeutic margin and efficacy[11] while focusing on unblemished targets. The current study focused on biological effects of a novel small molecule FCX in prostate cancer model. The antiproliferative efficacy of the compound in LNCaP cells was considered for selectivity to further explore the biological effects of the compound in prostate cancer.

It is well reported that the nuclear condensation/fragmentation is a common event in apoptosis.[12],[13] Morphological analysis with PI and Hoechst 333258 staining showed cell clumping and formation of apoptotic bodies characteristic of apoptosis in cells when treated with FCX. Cancer cells are prone to apoptotic deregulation, and with a potential to induce apoptosis, any drug can be considered as a novel for cancer treatment. When cancer cells undergo apoptosis, the prime event is the translocation of membrane phosphatidylserine (PS) from the inner side of the plasma membrane to the outer surface of the cell. Annexin V binds with high affinity to PS when exposed to the apoptotic cell surface. It also differentiates early and late phases of apoptosis taking into account the membrane integrity of the apoptotic cells.[14] The occurrence of dose dependence early, late, and total apoptosis of LNCaP cells in Annexin V assay with FCX treatment confirmed the apoptotic events of the compound.

There are studies to show that anticancer drugs arrest cells in the growth phase of the cell cycle and subsequently induce cell death by apoptosis.[15] It has been proven that sequence of events occurs as the damaged cells progress through the cell cycle arrest in G2/M phase which then passes through an aberrant mitosis to subsequently undergo apoptosis.[16] This type of apoptosis usually occurs as a later event in cell death resulting from a number of distinct pathways.[17] Certain agents are shown to cause DNA damage which results in stagnation of cells in G2/M phase before induced apoptosis.[18] The present results showed cell cycle arrest by FCX G2/M phase of LNCaP cell cycle. This cell cycle arrest was also associated with apoptosis-inducing property by the compound at higher concentrations suggesting the concentration-dependent efficacy of FCX in prostate cancer cells.

It is flagrant that pathway elucidation serves as a key point for further developments after preliminary preclinical regimens. The antiproliferative activity of FCX was more directed towards the androgen-dependent LNCaP cell when compared with the androgen-independent PC3 cells. To explore this, we used LNCaP prostate cancer cell line, containing a functional but mutated AR. The antiproliferative assay of LNCaP cells was carried out after incubating with desired concentrations of β-estradiol (E2), testosterone (T), and DHT for a period of 3 days followed by FCX treatment. LNCaP cells are androgen-sensitive human prostate adenocarcinoma cells and are hormonally responsive, shown by in vitro 5 alpha-DHT modulation of cell growth and acid phosphatase production.[19] When the steroid-responsive human LNCaP prostate cancer cell line, containing a functional but mutated AR, was used to monitor effects of β-estradiol (E2), testosterone (T), and DHT on cell proliferation, the cell proliferation was significantly stimulation by all four steroids. T and DHT demonstrating marked induction as compared to E2 induced a response which was in line with previously reported studies.[20],[21] Addition of FCX either alone or in the presence of these steroids shifted the GI50 of FCX markedly as shown in [Figure 5]. It was evident that all steroids showed protective effect in prostate cancer cells. It can, therefore, be hypothesized that FCX induces cytotoxicity and apoptosis, via ARs of LNCaP cells.

 > Conclusions Top

FCX has emerged as a potential lead candidate among the library of novel small molecules screened against prostate and colon cancer cells. The antiproliferative, apoptosis-inducing efficacy, and cell cycle changes of FCX demonstrated in vitro efficacy of the molecule in prostate cancer. Preliminary studies indicate the effect to take place through hormone-dependent effects. However, further evaluation of this molecule is needed to enumerate the complete pathway involved and develop it into a potential chemotherapeutic against prostate cancer.

Financial support and sponsorship

Authors acknowledge The Deanship of Scientific Research, King Khalid University, Abha, Saudi Arabia for their support (33/1439).

Conflicts of interest

There are no conflicts of interest.

 > References Top

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