IND-2, a Quinoline Derivative, Inhibits the Proliferation of Prostate Cancer Cells by Inducing Oxidative Stress, Apoptosis and Inhibiting Topoisomerase II

In men, prostate cancer (PC) is the most frequently diagnosed cancer, causing an estimated 375,000 deaths globally. Currently, existing therapies for the treatment of PC, notably metastatic cases, have limited efficacy due to drug resistance and problematic adverse effects. Therefore, it is imperative to discover and develop novel drugs for treating PC that are efficacious and do not produce intolerable adverse or toxic effects. Condensed quinolines are naturally occurring anticancer compounds. In this study, we determined the in vitro efficacy of IND-2 (4-chloro-2-methylpyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinolone) in the PC lines, PC-3 and DU-145. IND-2 significantly inhibited the proliferation of PC-3 and DU-145, with IC50 values of 3 µM and 3.5 µM, respectively. The incubation of PC-3 cells with 5 and 10 µM of IND-2 caused the loss of the mitochondrial membrane potential in PC-3 cells. Furthermore, IND-2, at 5 µM, increased the expression of cleaved caspase-3, cleaved caspase-7 and cleaved poly (ADP-ribose) polymerase (PARP). The incubation of PC-3 cells with 5 µM of IND-2 significantly decreased the expression of the apoptotic protein, B-cell lymphoma 2 (Bcl-2). Furthermore, 5 and 10 µM of IND-2 produced morphological changes in PC-3 cells characteristic of apoptosis. Interestingly, IND-2 (2.5, 5 and 10 µM) also induced mitotic catastrophe in PC-3 cells, characterized by the accumulation of multinuclei. The incubation of DU-145 cells with 1.25 and 5 μM of IND-2 significantly increased the levels of reactive oxygen species (ROS). Finally, IND-2, at 10 μM, inhibited the catalytic activity of topoisomerase IIα. Overall, our findings suggest that IND-2 could be a potential lead compound for the development of more efficacious compounds for the treatment of PC.


IND-2 Induces Apoptosis in PC-3 Prostate Cancer Cells
Apoptosis is a physiological process that is crucial for maintaining homeostasis in multicellular organisms [17,18]. However, numerous studies indicate that cancer cells can become resistant to certain anti-cancer drugs by evading apoptosis [19,20]. The induction of cellular apoptosis produces chromatin condensation, nuclear fragmentation, nuclear and cytoplasmic condensation, cellular rounding, membrane blebbing, ultrastructural modification of cytoplasmic organelles, loss of membrane integrity and finally, the formation of apoptotic bodies [17,21].

IND-2 Induces Apoptosis in PC-3 Prostate Cancer Cells
Apoptosis is a physiological process that is crucial for maintaining homeostasis in multicellular organisms [17,18]. However, numerous studies indicate that cancer cells can become resistant to certain anti-cancer drugs by evading apoptosis [19,20]. The induction of cellular apoptosis produces chromatin condensation, nuclear fragmentation, nuclear and cytoplasmic condensation, cellular rounding, membrane blebbing, ultrastructural modification of cytoplasmic organelles, loss of membrane integrity and finally, the formation of apoptotic bodies [17,21].
The incubation of PC-3 cells with 5 or 10 µM of IND-2 for 72 h induced shrinkage, rounding and the rupture of PC-3 cells (indicated by black arrows), as shown in Figure 1e. Live cell imaging indicated that 5 or 10 µM of IND-2 increased the number of non-viable or dead PC-3 cells, compared to PC cells incubated with vehicle control (Figure 1f).

IND-2 Significantly Alters the Mitochondrial Membrane Potential of PC-3 Cells
Mitochondria play a significant role in the regulation of apoptosis [22]. A loss of mitochondrial membrane potential, following the activation of executioner caspases, is one of the earliest steps in apoptosis [23]. Therefore, we determined the effect of IND-2 on the mitochondrial membrane potential. This was done using the tetramethyl rhodamine ester (TMRE) assay. TMRE is a red fluorescent dye that readily accumulates in viable mitochondria due to their negative charge [24]. IND-2, at 5 µM, significantly decreased (p < 0.001) fluorescent intensity in PC-3 cells, compared to cells incubated with vehicle, indicating the loss of viable mitochondria due to the loss of mitochondrial membrane potential (∆Ψm) (Figure 2a,b) Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) is an inducer of plasma membrane depolarization in many types of cells from different species, which disrupts the mitochondrial membrane potential [25]. Consequently, we used this compound as a positive control (Figure 2a,b). Our findings suggest that IND-2 produces, in part, its anti-cancer efficacy by decreasing the number of viable mitochondria and the loss of the mitochondria membrane potential, thus increasing the likelihood of apoptosis in PC-3 cells. These data were further validated using another dye, JC-1. The decrease in the ratio of red to green fluorescence for JC-1 indicates a loss of mitochondrial membrane potential. In PC-3 cells incubated with vehicle the mitochondria appear red with minimal green fluorescence, whereas in cells incubated with IND-2 and the positive control compound, FCCP, the level of green fluorescence increased and the level of red fluorescence decreased, indicating that IND-2 causes the loss of the mitochondrial membrane potential.

Effect of IND-2 on the Cell Cycle of PC-3 Cells
The majority of clinically approved chemotherapeutic drugs induce cancer cell death by apoptosis [26][27][28]. Furthermore, anti-cancer drugs produce cell cycle arrest and inhibit the checkpoint signaling pathways in cancer cells [29]. Thus, we determined the effect of IND-2 on the cell cycle in PC-3 cells. The incubation of PC-3 cells with vehicle (negative control) produced the following distribution in the different phases of the cell cycle: 38.66% in the sub-G0 phase, 36.29% in the G0/G1 phase, 19.87% in the S phase and 2.06% in the G2 phase. (Figure 3a). After the incubation of PC-3 cells with 5 and 10 µM of IND-2, the population of cells in the G2 phase increased to 5.26% and 15.06%, respectively, compared to cells incubated with vehicle. Overall, our results indicated that IND-2 produced significant cell cycle arrest in the G2 phase ( Figure 3b).

IND-2 Induces Nuclear Condensation and Mitotic Catastrophe in PC-3 Cells
Morphologically, apoptosis induces cellular alterations, characterized by condensation and fragmentation of nuclei and cells and the fragmentation of chromosomal DNA into nucleosomal units [20,30]. Cell shrinkage occurs at the onset of apoptosis, followed by the condensation of the nucleus and nuclear chromatin that become highly delineated masses marginated against the nuclear membranes [17]. We determined the effects of IND-2 on the nuclear morphology of PC-3 cells. The incubation of PC-3 cells with Hoescht dye, followed by incubation with 5 and 10 µM of IND-2, indicated the presence of nuclear condensation and fragmentation (Figure 4a). In contrast, incubating PC-3 cells with vehicle did not produce nuclear condensation and fragmentation. (Figure 4a).      micronuclei which induces apoptotic morphology [34]. The incubation of PC-3 cells with 2.5 µM of IND-2 produced large cells with multiple micronuclei, which did not occur in cells incubated with vehicle ( Figure 4b). The incubation of PC-3 cells with 5 and 10 µM of IND-2 produces several multinucleated cells similar to that of 2.5 µM of IND-2. (Figure  4b). The induction of MC has been reported to overcome resistance of cancer cells to drugs that induce apoptosis [35]. Therefore, the induction of MC by IND-2 could overcome resistance to apoptosis in cancer cells [35].  Mitotic catastrophe (MC) occurs when cells fail to undergo mitosis, causing the cells to undergo apoptosis, non-apoptotic death or senescence [31]. The 2012 Nomenclature Committee on Cell Death (NCCD) stated that MC is activated during the M phase by damaging or disrupting the mitotic apparatus, producing mitotic arrest that causes cell death or senescence [31]. MC has been postulated to occur before the onset of apoptosis [31].
Morphologically, MC is characterized by the formation of micronuclei that arise from the nuclear envelope around mis-segregated chromosomal clusters that are one-third of the size of a normal nucleus [31][32][33]. A number of anti-cancer drugs, including cisplatin, taxanes, doxorubicin and etoposide, have been reported to induce cancer cell death by producing MC [31]. Furthermore, MC produces the formation of large cells with multiple micronuclei which induces apoptotic morphology [34]. The incubation of PC-3 cells with 2.5 µM of IND-2 produced large cells with multiple micronuclei, which did not occur in cells incubated with vehicle ( Figure 4b). The incubation of PC-3 cells with 5 and 10 µM of IND-2 produces several multinucleated cells similar to that of 2.5 µM of IND-2. (Figure 4b).
The induction of MC has been reported to overcome resistance of cancer cells to drugs that induce apoptosis [35]. Therefore, the induction of MC by IND-2 could overcome resistance to apoptosis in cancer cells [35].

IND-2 Activates the Apoptotic Pathway and Induces DNA Fragmentation
Caspases are an evolutionarily conserved family of cysteine-dependent endoproteases that play an essential role in the induction of apoptosis [36][37][38]. For example, caspase-3 and caspase-7, known as the executioner caspases, produce the morphological changes associated with the induction of apoptosis [36]. The activation of caspase-3 and caspase-7 increases the expression of the cleaved forms of caspase-3 and caspase-7, resulting in induction of apoptosis [39]. We determined the expression levels of apoptotic proteins hallmark characteristic of apoptotic cell death [40,41]. (Figure 5a,c). The Bcl-2 protein family can produce apoptosis by altering MOMP and the subsequent activation of downstream caspase-3 and caspase-7 [42,43]. The Bcl-2 gene is overexpressed in a wide variety of human cancers [44]. Furthermore, the overexpression of the Bcl-2 gene is positively correlated with anticancer drug resistance and efficacy of many clinically used anti-cancer drugs is decreased [44]. Bcl-2 produces anticancer drug resistance by decreasing the probability of apoptosis [44]. Thus, the expression of the Bcl-2 gene and protein represent a major therapeutic target for the design of anti-cancer drugs. The incubation of PC-3 cells with 5 µM of IND-2 decreased the levels of Bcl-2, compared to cells incubated with vehicle (Figure 5a,c). The proteins, Bcl-2 Antagonist/Killer 1 (BAK) and Bcl-Associated X protein, (BAX), are pro-apoptotic proteins that increase the probability of apoptosis [45]. The expression of the BAK gene in PC-3 cells was not significantly altered by 1 or 5 µM of IND-2, compared to cells incubated with vehicle. Interestingly, we saw BAX downregulation after incubation with IND-2 that was comparable to that of the positive control. The loss of BAX and Bcl-2 expression has not been reported before and it remains to be determined what mechanism produces this dual loss. It appears, however, that BAX forms heterodimers with the protein, Myeloid Cell Leukemia 1 (MCL-1), which prevents apoptosis from occurring [46,47]. It remains to be elucidated how IND-2 modulates MCL-1 and regulates Bcl-2 and BAX. Based on our in vitro results, IND-2 increases the probability of cellular apoptosis by altering the expression of several key apoptotic proteins (Figure 5a,c).
DNA fragmentation is one of the hallmarks of apoptotic cell death [48,49]. Therefore, we conducted experiments to determine if IND-2 produces DNA fragmentation. The incubation of PC-3 cells for 8 or 12 h with vehicle (negative control) did not produce DNA fragmentation (Figure 5b). However, the incubation of cells with 1 or 5 µM of IND-2 for 8 or 12 h significantly increased DNA fragmentation compared to cells incubated with vehicle ( Figure 5b).

IND-2 Induces Oxidative Stress in DU-145 Cells
Cancer cells have an antioxidant system that protects them from damage due to the presence of low-moderate levels of intracellular reactive oxygen species (ROS) [50,51]. However, high levels of ROS can surmount the antioxidant system, causing cancer cell death [50]. It has been hypothesized that chemotherapeutic amplification of ROS levels in cancer cells should increase the probability of cell death [52]. Several clinically approved chemotherapeutic drugs can induce oxidative stress in cancer cells [51]. Specifically, anthracyclines, such as daunorubicin, doxorubicin, and epirubicin, increase ROS production in cancer cells [52,53]. Other drugs, such as alkylating agents, platinum coordination complexes, camptothecins, arsenic-based drugs and topoisomerase inhibitors, also induce ROS [52]. As ROS levels increase, the p53 enzyme system becomes activated, and apoptosis is upregulated by increasing transcription of pro-apoptotic proteins, such as BAX and BAK, and by decreasing the transcription of the pro-survival proteins, such as Bcl-2, B-cell lymphoma extra-large (BCL-XL) and MCL-1 [54]. Furthermore, p53, a tumor suppressing protein, increases mitochondrial membrane permeabilization, allowing cytochrome c to be released, an apoptotic molecule, thus increasing the probability of cell death [55]. As a result, we determined whether IND-2 affected ROS levels in DU-145 cells. The ROS levels were determined using 2, 7-dichlorodihydrofluorescein diacetate (H2DCFDA), which is hydrolyzed by esterase enzymes when entering cells, resulting in the formation of H2DCF, which is then oxidized by ROS within the cells into dichlorofluorescein (DCF) [56]. DCF is a highly fluorescent compound and its fluorescence intensity is positively correlated with the intracellular levels of ROS levels [56]. The incubation of DU-145 with 5 µM of IND-2 and 1 µM of paclitaxel produced a significantly greater level of fluorescence, compared to the vehicle control; p < 0.001 (Figure 6a,b). Our results are consistent with previously reported findings that paclitaxel significantly increased intracellular ROS levels in cancer cells [57,58].

IND-2 Significantly Inhibits the In Vitro Invasiveness and the Migration Potential of PC-3 Cells
The metastatic progression of cancer is the primary cause of death in patients [59]. Therefore, it is important to develop anti-cancer drugs that prevent cancer cell migration and invasion. To determine whether IND-2 has anti-metastatic efficacy in PC-3 cells, we used an in vitro invasive assay, known as the wound healing assay. IND-2, at 10 µM, significantly inhibited the migration and invasion of PC-3 cells, as early as 24 h, compared to cells incubated with vehicle (Figure 7a,c). The PC-3 cells failed to invade and cover the wound after incubation with 10 µM of IND-2, as compared to cells incubated with vehicle (Figure 7a,c). Furthermore, the wound width remained the same up to 72 h of incubation, starting from 8 h post-incubation (p < 0.001) (Figure 7a,c).

IND-2 Induces Oxidative Stress in DU-145 Cells
Cancer cells have an antioxidant system that protects them from damage due to the presence of low-moderate levels of intracellular reactive oxygen species (ROS) [50,51]. However, high levels of ROS can surmount the antioxidant system, causing cancer cell death [50]. It has been hypothesized that chemotherapeutic amplification of ROS levels in cancer cells should increase the probability of cell death [52]. Several clinically approved chemotherapeutic drugs can induce oxidative stress in cancer cells [51]. Specifically, an- the formation of H2DCF, which is then oxidized by ROS within the cells into dichlorofluorescein (DCF) [56]. DCF is a highly fluorescent compound and its fluorescence intensity is positively correlated with the intracellular levels of ROS levels [56]. The incubation of DU-145 with 5 µM of IND-2 and 1 µM of paclitaxel produced a significantly greater level of fluorescence, compared to the vehicle control; p < 0.001 (Figure 6a, b). Our results are consistent with previously reported findings that paclitaxel significantly increased intracellular ROS levels in cancer cells [57,58].

IND-2 Significantly Inhibits the In Vitro Invasiveness and the Migration Potential of PC-3 Cells
The metastatic progression of cancer is the primary cause of death in patients [59]. Therefore, it is important to develop anti-cancer drugs that prevent cancer cell migration and invasion. To determine whether IND-2 has anti-metastatic efficacy in PC-3 cells, we used an in vitro invasive assay, known as the wound healing assay. IND-2, at 10 µM, significantly inhibited the migration and invasion of PC-3 cells, as early as 24 h, compared to cells incubated with vehicle (Figure 7a, c). The PC-3 cells failed to invade and cover the wound after incubation with 10 µM of IND-2, as compared to cells incubated with vehicle (Figure 7a, c). Furthermore, the wound width remained the same up to 72 h of incubation, starting from 8 h post-incubation (p < 0.001) (Figure 7a,c).
One of the major hallmarks of cancer metastasis is the epithelial to mesenchymal transition (EMT) [60]. The EMT trans-differentiation process confers the transformed One of the major hallmarks of cancer metastasis is the epithelial to mesenchymal transition (EMT) [60]. The EMT trans-differentiation process confers the transformed epithelial cells with the capacity to invade, resist stress and disseminate [60,61]. In addition, several studies have shown that the Wingless-related integration site (Wnt)/β-catenin signaling pathway may play a key role in EMT [62]. Therefore, we determined the expression of EMT and Wnt/β-catenin markers in PC-3 cells incubated with IND-2. The incubation of PC-3 cells with 5 µM of IND-2 significantly decreased the expression of the mesenchymal marker, N-cadherin and increased the expression of the epithelial marker, E-cadherin, compared to the cells incubated with vehicle ( Figure 7b). In addition, IND-2 significantly decreased the expression of Dishevelled Segment Polarity Protein 3 (DVL3), a key regulator of the Wnt/βcatenin pathway (Figure 7b) [63]. β-catenin is a downstream effector of the Wnt/β-catenin pathway [63]. High levels of β-catenin have been reported to increase tumorigenesis [63]. β-catenin induces tumorigenesis by the transactivation of its downstream target genes, cyclin-D1 and C-myc [63]. IND-2, at 5 µM, induced the fragmentation of β-catenin, as indicated by western blotting (Figure 7b), which did not occur in cells incubated with vehicle ( Figure 7b). The fragmentation of β-catenin primarily results from activation of apoptosis and plays a role in mediating the anti-proliferative and anti-metastatic efficacy in PC-3 cells [64]. Cyclin B1 is a protein that regulates the initiation of mitosis [65]. The overexpression of cyclin B1 is negatively correlated with a poor prognosis in different types of cancer and is involved in tumorigenesis [65]. Thus, decreasing the levels of cyclin B1 could be a potential approach for treating cancer. sults from activation of apoptosis and plays a role in mediating the anti-proliferative and anti-metastatic efficacy in PC-3 cells [64]. Cyclin B1 is a protein that regulates the initiation of mitosis [65]. The overexpression of cyclin B1 is negatively correlated with a poor prognosis in different types of cancer and is involved in tumorigenesis [65]. Thus, decreasing the levels of cyclin B1 could be a potential approach for treating cancer. The incubation of PC-3 cells with 5 µM of IND-2 significantly decreased the expression of cyclin B1 ( Figure  7b). Thus, IND-2 has a significant impact on the Wnt/β-catenin signaling pathway, as well as the expression of several EMT markers.

IND-2 Inhibits Topoisomerase II Activity
DNA topoisomerases are enzymes that prevent DNA supercoiling during replication, transcription, recombination and chromatin reorganization [66,67]. Because of the essential roles of these enzymes in the maintenance of cell function, topoisomerases have become targets for the development of anticancer drugs [66]. Since IND-2 shares structural similarity with the quinoline scaffold of many novel topoisomerase II inhibitors [68,69], we conducted a topoisomerase assay to determine if IND-2 inhibits topoisomerase II. IND-2 at 50 and 100 µM, significantly inhibited topoisomerase II activity, compared to cells incubated with vehicle ( Figure 8). Decatenation is a critical in vivo reaction of DNA topoisomerases and is an indicator of topoisomerase activity during DNA replication [68] Kinetoplast DNA is frequently used as a substrate in the decatenation assay [70]. In the kinetoplast DNA decatenation assay, IND-2, at 50 and 100 µM, significantly inhibited the activity of Topo IIα. This is in contrast to etoposide, which traps Topo IIα covalently bound to the cleaved DNA strands, inhibiting DNA synthesis [71]. IND-2, at 50 and 100 µM, inhibited topoisomerase IIα from accessing the substrate, kinetoplast DNA (Figure 8). The absence of the decatenated products of topoisomerase IIα, nicked open circular DNA and relaxed circular DNA, after incubation with 50 and 100 µM of IND-2, suggests that IND-2 inhibits the catalytic activity of topoisomerase IIα (Figure 8). Further studies, such as surface plasmon resonance kinetoplast DNA decatenation assay, IND-2, at 50 and 100 µM, significantly inhibited th activity of Topo IIα. This is in contrast to etoposide, which traps Topo IIα covalentl bound to the cleaved DNA strands, inhibiting DNA synthesis [71]. IND-2, at 50 and 10 µM, inhibited topoisomerase IIα from accessing the substrate, kinetoplast DNA (Figur 8). The absence of the decatenated products of topoisomerase IIα, nicked open circula DNA and relaxed circular DNA, after incubation with 50 and 100 µM of IND-2, sugges that IND-2 inhibits the catalytic activity of topoisomerase IIα (Figure 8). Further studie such as surface plasmon resonance (SPR) and binding studies, will be required to dete mine if topoisomerase II is a possible target for IND-2.

Chemistry: Synthesis and Characterization of IND-2
The procedure for the synthesis and the analytical data for the compound (IND-2 ha been previously reported [14]).

Chemistry: Synthesis and Characterization of IND-2
The procedure for the synthesis and the analytical data for the compound (IND-2 has been previously reported [14]).

Cell Line and Culture Conditions
The PC cells, DU-145 and PC-3, were a gift from the late Dr. Gary Kruh, University of Illinois at Chicago. These cells were grown as an adherent monolayer in a culture flask containing DMEM media that was supplemented with 4.5 g of glucose, 10% fetal bovine Life 2022, 12, 1879 12 of 18 serum (FBS) and 1% penicillin/streptomycin (in an incubator at 37 • C with 5% CO 2 and a relative humidity of 95%.

Cytotoxicity Assay
A cell cytotoxicity assay using the MTT assay was performed on PC cells using previously described protocols [26,72]. Briefly, the DU-145 cells were seeded in 96-well plates at a density of 3000 cells per well, and PC-3 cells were seeded in 96-well plates at a density of 2000 cells per well. Immediately following the culturing of the PC cells for 24 h, the cells were incubated for a second 72 h period with IND-2 (at 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, or 10 µM) or vehicle (DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin; control) or the test compound (1-10 µM). Immediately following incubation of the test compound in each well, 20 µL of a solution of MTT reagent at a concentration of 4 mg/mL was added to each well and the cells were incubated for 3 h at 37 degrees Celsius. The media was aspirated, and the formazan crystals that formed were dispersed in 150 µL of DMSO. BioTek™ Synergy™ H1Multi-Mode Reader (Winooski, VT, USA) was used to analyze the 96-well plates at a wavelength of 570 nm.

Colony Formation Assay
The effect of IND-2 on the proliferation of PC cells was determined by measuring the colony formation rate of PC-3 cells after incubation with IND-2. In a 6 well plate, the PC cells were seeded at a density of 250 cells/well and allowed to attach overnight. The following day, cells were incubated with IND-2 (2.5 and 10 µM) and this was done every 3 days for 14 days. On the 14th day, the media was aspirated, and the cells were fixed with methanol. Finally, the cells were incubated with 0.1% crystal violet dye for 20-30 min. The cells were washed vigorously three times with PBS to remove the excess stain and images of the colonies were using the BioTek Citation 7™.

Live Cell Imaging and Morphological Analysis
The effect of IND-2 on the morphology of PC-3 cells was determined by live cell imaging using BioTek™ Synergy™. Briefly, PC-3 cells were seeded at a density of 3000 cells/well in a 96 well plate. The following day, the cells were incubated with vehicle (negative control) and IND-2 (1.25, 2.5, 5 and 10 µM) and transferred to BioTek Cytation 7™ for live cell imaging. The images were captured at 20-fold magnification for a period of 72 h and the changes in the cell confluence over time was determined based on the images captured at 4-fold magnification.
3.2.5. Cell Cycle Checkpoint Analysis using Fluorescence-Activated Cell Sorting (FACS) As described previously [73], a flow cytometric analysis of the cell cycle was performed using propidium iodide (PI) staining to determine the distribution of the cells at each phase of the cell cycle. PC-3 cells were seeded in a 6-well plate at a density of 250,000 cells/well. In a 6-well plate, PC-3 cells were seeded at a density of 250,000 cells per well. Immediately after incubation, the cells were incubated with vehicle or IND-2 at 2.5, and 10 µM for 24 h. Subsequently, the cells were trypsinized with 0.05% trypsin and 2.21 mM EDTA, washed once in phosphate-buffered saline, and resuspended in 1 mL of ice-cold PBS. For staining the cells, 10 µL of the 500 µg/mL PI stock solution was applied. Cells were then incubated on ice for 15 min. The BD FACS Canto™ flow cytometer (BD Biosciences, Becton-Dickinson, San Jose, CA, USA) was used to determine the distribution of the cells in the cell cycle phases (SubG1, G1, S, G2). The data was analyzed using FCS Express 7 cytometry (De Novo software, Pasadena, CA, USA).

Determination of Reactive Oxygen Species (ROS) Levels
A 96-well plate containing PC-3 cells was seeded at a density of 3000 cells per well and incubated overnight. The adherent cells were treated with either vehicle (DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin; control), IND-2 (1.25 Life 2022, 12, 1879 13 of 18 and 5 µM) or the positive control, paclitaxel (0.5 µM) for 24 h. Subsequently, the cells were incubated with 3 µM of DCFDA (2 ,7 -dichlorodihydrofluorescein diacetate) for 30 min, followed by gentle washing with ice cold PBS three times. Based on the level of fluorescence generated by the oxidized DCFDA dye, the level of ROS was quantified using BioTek Cytation7™ at a 20-fold magnification.

Determination of Mitotic Catastrophe
The PC-3 cells were cultured into 6-well plates with microscopic cover slips at a density of 200,000 cells per well and incubated overnight. After adhering for 12 h, the cells were incubated with vehicle (DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin; control) or 2.5 and 10 µM of IND-2. Following incubation for 24 and 48 h, the cells were fixed with 4% paraformaldehyde (1 mL/well) for 15 min. The cells were then gently washed twice with PBS Microscopic coverslips were removed and mounted on glass slides with the cells facing the glass slide in one drop of DAPI (4 ,6-diamidino-2-phenylindol) at 1 µM. A fluorescence image of the stained nuclei was obtained using the blue channel in BioTek Cytation 7 TM at a 20-fold magnification (DAPI has a maximum absorption wavelength of 358 nm and a maximum emission wavelength of 461 nm).

Detection of Nuclear Condensation
The nuclear condensation of PC-3 cells following incubation with IND-2 (2.5 and 10 µM) was detected using Hoescht dye, which stains the DNA in the cell nucleus. Briefly, the PC-3 cells were seeded at a density of 3000 cells per well in a 96-well plate. After overnight incubation, the cells were exposed with vehicle (DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin; control) and IND-2 (2.5 and 10 µM). After 24 h of incubation, 1 µM of Hoescht dye was added to the wells and incubated for 20 min. The cells were then imaged on the DAPI channel at 20-fold magnification.

Determination of Mitochondrial Membrane Potential
The induction of apoptosis and changes in mitochondrial membrane potential in PC-3 cells were analyzed using Tetramethylrhodamine, ethyl ester (TMRE) dye. TMRE stains active mitochondria and produces red fluorescence when taken up by active live cells [24]. Briefly, PC-3 cells were seeded at a density of 3000 cells per well in a 96 well plate. After overnight incubation, the cells were exposed with IND-2 at 5 µM and incubated for another 24 h. These cells were then incubated with the positive control, trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP 50 µM) for 1 h. After incubation with IND-2 and FCCP, 1 µL of TMRE dye was added to each well and the cells were imaged on a Texas Red channel using a BioTek Cytation 7™ at 20× magnification. The fluorescence intensity was quantified by measuring the mean fluorescence intensity at excitation/emission wavelength of 549/575 nm.
The loss of mitochondrial membrane potential was further validated by using the dye, JC-1. Briefly, PC-3 cells were seeded in a 96 well plate at a eedng density of 2000 cells/well. The following day, cells were incubated with IND-2 (5 µM) for 24 h. The following day, cells were incubated with CCCP (50 µM) for 4 h followed by JC-1 (2 µM) and Hoescht (3 µg/mL) and incubated for 30 min. Cells were washed with PBS and imaged using Biotek Cytation 7™ at 20× magnification.

DNA Fragmentation
The DNA extraction and fragmentation assay was used to determine if IND-2 induced apoptosis, as DNA fragmentation precedes the morphological changes produced by apoptosis. Approximately 2 × 106 cells were seeded in 6 mm Petri dishes. The following day, the cells were incubated with vehicle (negative control) and IND-2 (1 and 5 µM). The cells were trypsinzed 8 or 12 h later and washed once with PBS. Additionally, 100 µL of Tris-EDTA (TE) buffer (pH 7.4), with 2% SDS was added to the pellet, followed by mixing and vortexing. Subsequently, the solution was centrifuged at 15,000 rpm for 10 min at 4 • C. DNA was isolated from the supernatant and analyzed using UV (Ultraviolet) spectroscopy by recording the absorbance at 260, 280, and 230 nanometers. Using a ratio of A260/A280, the purity of the isolated DNA was determined. Nucleic acids have an absorbance maximum at 260 nm, and the ratio between this absorbance and the absorbance at 280 nm is used as a measure of purity [76]. After DNA was isolated, it was loaded onto a 1.5% agarose gel and run at 35 V for 6-8 h. An UV light was used to visualize the DNA smear to determine if it was fragmented [77].

Wound Healing Assay
The wound healing assay, also known as the scratch assay, was used to evaluate IND-2's efficacy to prevent invasion and metastasis of the prostate cancer cells. Briefly, PC-3 cells were seeded at a density of 10,000 cells per well in a 96-well plate and allowed to attach overnight. The following day, the cells were observed to determine if they had reached at least 95% confluency and formed a monolayer. If the cells were determined to have formed a monolayer, a scratch was made in the center, using the Incucyte ® 96-Well Woundmaker Tool. The cells were immediately incubated with IND-2 (2.5 and 10 µM) and transferred to the BioTek Cytation 7™ to capture live images from (0, 8, 24, 48, 72 h) using the brightfield channel at 4-fold magnification.

Topoisomerase II Inhibitory Assay
The assay was done according to the manufacturer's instructions. Briefly, 100 ng of catenated kinetoplast DNA, 8 units of topoisomerase IIα, together with the test compounds, were incubated for 30 min at 37 • C in 50 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 10 mmol/L MgCl 2 , 0.5 mmol/L Dithiothreitol, 30µg/mL BSA, and 2 mmol/L ATP in final volume of 20 µL of the reaction mixture. The reaction was stopped by the addition of 2 uL of the stop solution (10% SDS) and incubated with 50 µg/mL proteinase K for 15 min at 37 • C. kDNA was electrophoresed on a EB containing 1% agarose gel to separate topoisomers and analyzed using a gel documentation system, G:BOX CHEMI XX9 (Syngene, Frederick, MD, USA).

Statistical Analysis
Data analysis and data representation were performed utilizing the GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). The IC50 values obtained from the MTT assay were determined using an unpaired t-test with Welch's correction. Cell cycle distributions and Western blot protein expression quantification were analyzed with two-way ANOVA followed by Bonferroni's post hoc analysis. In the ROS assay, green fluorescence intensity was analyzed using an unpaired t-test with Welch's correction.

Conclusions
Our results indicated that, in vitro, IND-2 significantly inhibited the proliferation of PC cancer cells DU-145 and PC-3 and the formation of PC-3 colonies. Furthermore, IND-2 had anti-metastatic efficacy, based on its decrease in the expression of the EMT proteins, DVL3 and Cyclin B1, and its decrease in the invasiveness and migration of PC-3 cells. Our mechanistic experiments indicated that based on the MTT and colony forming assays and live cell imaging, IND-2 significantly decreased the proliferation of PC-3 and DU-145 cells. Additional experiments indicated that IND-2 could produce its anti-cancer efficacy by inducing oxidative stress, decreasing the mitochondrial membrane potential and inhibiting metastasis by altering the levels of Wnt 5a/b, DVL3, E-cadherin and cyclin B1. Furthermore, SwissADME, an in-silico web tool, has indicated that IND-2 has favorable physicochemical properties, drug-like characteristics and high bioavailability (Supplementary Figure S1) [78]. Overall, our results suggest that IND-2 may represent a promising lead compound for the development of compounds that inhibit the growth, proliferation and metastasis of PC cells.