Investigation of the Effect of Phenylboronic Acid on Androgen-Dependent (LNCaP) and Androgen-Independent (PC3) Prostate Cancer Cells via MAP Kinases by 2D and 3D Culture Methods

doi:10.21203/rs.3.rs-3811631/v1

Download PDF

Research Article

Investigation of the Effect of Phenylboronic Acid on Androgen-Dependent (LNCaP) and Androgen-Independent (PC3) Prostate Cancer Cells via MAP Kinases by 2D and 3D Culture Methods

https://doi.org/10.21203/rs.3.rs-3811631/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

Background

The castration process is able to regress prostate cancer due to its dependence on androgen. After castration, the disease could progress androgen independently. In our study, two prostate cancer cell lines PC3, LNCaP, and normal prostatic epithelial cell line RWPE-1 were used. PBA an essential compound found in nature, was selected as a chemotherapeutic to examine the effects of microtubule-targeted therapy in human prostate cancer. Colchicine, which belongs to the same class of chemotherapeutics, was included in the study as a positive control treatment. The aim of this study examine the cytotoxic effect of PBA on LNCaP, PC-3, and RWPE-1 cells with two different cell culture methods.

Methods

The IC₅₀ values treated to the cells following the viability analyses were performed for PBA and Colchicine in 2D and 3D culture models. Colony formation, proliferation, and migration analyses were performed on prostate cancer cells, and chemotherapeutics’s effects were compared.

Results

In both cancer cell lines, 48 hours of PBA treatment inhibited migration greater than Colchicine. Colony formation analysis showed that the 24 hours PBA treatment prevented the formation. In addition, it was determined that PBA caused a decrease in proliferation parameters in both culture models. The MAPK cellular response induced by PBA was examined by immunofluorescence intensity analysis of kinase proteins of the MAPK pathway, where statistically significant differences were observed between the groups. ERK expression ratio varied in two culture methods, chemotherapeutics, and treatment times. In the 2D culture model, 24 hours of PBA treatment caused a decrease in JNK expression in PC3 and LNCaP cells. Both chemotherapeutic treatments resulted in an increase in p38 expression ratio in PC3 spheroids. On the semi-thin sections, the morphological deformation effect of PBA on cancer cells was pronounced. Morphological defects caused by PBA were first visualized in this study at the ultrastructural level.

Conclusion

Antimitotic chemotherapeutics may trigger different metabolic responses and also divergences in the signaling mechanisms within different cells. PBA has an anticancer effect potential including inhibiting proliferation and migration. The lower toxicity of PBA on RWPE-1 is remarkable for being a potential chemotherapeutic option in future research.

During a lifetime, one out of every eight men is diagnosed with prostate cancer, according to the American Cancer Community [1]. Castration is generally preferred for anticarcinogenic treatment, especially for androgen-dependent cases. Nonetheless, cancer could come over castration and continue as androgen-independent through development and metastasis[2]. Due to these situations, prostate cancer progresses in two phases: androgen dependent (castration-dependent) and androgen-independent (castration-independent) [3]. The difference between human and rodent prostates limits the examination of prostate cancer in model organisms [4].

Because of the ethical limitations and physiological barriers to obtaining human samples, human-origin research are restrictive. Hence, most clinical reports are insufficient for statistical analysis and the repeating principle of research perspective despite the majority of them in literature [5–7]. On the other hand human-originated cancer cells are preferred for providing more information about human cancer metabolism [4]. Two-dimensional cell culture research has also drawn attention in the literature but is also limited in clinical reports because it does not contain a natural tumorogenic environment. Otherwise, three-dimensional cancer cell culture experiments show closer results to those in vivo [8]. Drug resistance in vivo is the most common characteristic of cancer and is only seen in three-dimensional culture methods, not in two-dimensional methods [9, 10]. Additionally, apoptotic responses differ between two-dimensional and three-dimensional culture methods [11]. Riedel et al. demonstrated differences between two-dimensional and three-dimensional culture methods including phenotype, (adenosine triphosphate) ATP mechanism, decreasing cell cycle progression in spheroids, decreasing signaling of (Ak strain transforming)AKT– (mammalian target of rapamycin) mTOR–S6 pathway in spheroids, and triggered (extracellular signal-regulated kinase) ERK phosphorylation only in 2D culture model while it is reduced following the AKT inhibition in 3D cell culture model [12].

Organic and natural compounds are preferred for anticarcinogenic therapies because of their possible lower toxicity. Phenylboronic acid (PBA) is a natural compound that people consume daily in boron form [13, 14]. It belongs to the boric acid family and has been noted in the literature to have an anticarcinogenic effect on prostate and mammary carcinoma [15–17]. A study conducted in an area that contains boron in drinking water shows an infrequent prostate cancer incidence [18]. Few researchers have addressed the question about the anticarcinogenic effect of PBA. It was published that PBA decreases the prostate-specific antigen (PSA) level in prostatic cancer cells [17]. Another study showed the decreasing proliferative activity of LNCaP, PC3, and DU-145 cells depending on the dose of PBA treatment [19].

Chemotherapeutics could affect the cytoskeleton of cancer cells to block mitosis by inhibiting the polymerization or depolymerization of subunits. They are classified according to their mechanism of action. It has been shown that PBA inhibits the cytoskeleton as a microtubule destabilizing agent due to its tubulin polymerization inhibitor effect [20, 21]. Several immunofluorescence studies demonstrated a destroyed cytoskeleton after PBA treatment of cancer cells [22, 23]

Microtubule-targeted therapeutics trigger apoptosis by the (mitogen-activated protein kinase) MAPK signaling pathway, which is involved in survival, proliferation, and apoptosis metabolism [24]. It has been reported that after numerous microtubule inhibitor treatments, JNK(c-Jun N-terminal kinase) was activated, ERK was not affected, and only Taxol treatment resulted in increased (p38 mitogen-activated protein kinase-p38 MAPK, termed as p38) p38 expression [25, 26]. Barranco and Eckhert reported that boric acid shows its anticarcinogenic effect via the Raf/MEK/ERK1/2 MAPK signaling pathway in DU-145 cells [27]. However, the underlying mechanisms of the anticarcinogenic effect of PBA are not entirely understood.

There is no research on the anticarcinogenic effect of PBA on prostate cancer cells representing castration-dependent (LNCaP) and castration-independent (PC3) cell lines and a normal prostatic epithelial cell line (RWPE-1). In this study, we compared the cytotoxic effect of PBA on LNCaP, PC-3, and normal epithelial RWPE-1 cells and two different cell culture methods. The changes in ERK, JNK, and p-38 expression ratios in PBA-treated LNCaP and PC-3 cells were examined, and the possible molecular mechanism underlying the anticarcinogenic effect of PBA was hypothesized.

Cell culture

One of the three human prostate cancer cell lines is PC3 (CRL-1435, ATCC), which was obtained from our department stock. Another cell line, LNCaP (CRL-1740, ATCC), was donated by Prof. Dr. Engin Ulukaya from Istinye University. The third cell line, RWPE-1, was donated by Dr. Yasemin Yozgat from Istanbul Medipol University. PC3 and LNCaP cell lines were cultured in RPMI 1640 with L-glutamine (RPMI-A, Capricorn) supplemented with 10% FBS (FB-1001/500, Biosera) and antibiotic-antimycotic (XC-A4110/20, Biosera) at 37°C and 5% CO₂. The RWPE-1 cell line was cultured in keratinocyte serum-free media (17005042, Thermo Fisher Scientific) supplemented with human recombinant epidermal growth factor (EGF1-53) and bovine pituitary extract (BPE) (Keratinocyte-SFM Supplement 37000015, Thermo Fisher Scientific) without antibiotic-antimycotic at 37°C and 5% CO₂.

2D cell culture

When cells reached 70-80% confluency in T25 flasks, they were trypsinized (5 minutes for PC3, 3 minutes for LNCaP) and collected in tubes after centrifugation at 1500 rpm for 5 minutes. Following cell counting, the cells were split into new wells for the 3D culture method and/or seeded in IBIDI 12-well slides for the continuing 2D culture process. Due to the slower metabolism, LNCaP cells were seeded at 1.2x10⁴ cells/200 µl, RWPE-1 cells were seeded at 2x10⁴ cells/200 µl, and PC3 cells were seeded at 1x10⁴ cells/200 µl on slides in an appropriate culture medium.

3D cell culture

Each well of 96-well plates was covered with 100 µl of 3% warm agarose gel solution to obtain a ‘U’-shaped well bottom. PC3 cells were seeded at 1x10⁴ cells/200 µl, and LNCaP cells were seeded at 3.5x10⁴ cells/200 µl into each well. After seeding, the plates containing LNCaP cells were centrifuged for 3 minutes at 1200 rpm [28]. Spheroids grew to 350-400 µm diameters in 5 days by refreshing media to half volume every other day. Spheroids with different diameters were excluded from the experiments. Because of the slower metabolism of RWPE-1 cells, it is difficult to boost them to construct a spheroid structure. Any triggering manipulation brings a carcinogenic effect on these epithelial cells, which means changing their normal metabolism functions. On the other hand, the monolayer PBA viability analysis showed lower toxicity in RWPE-1 cells. Therefore, RWPE-1 cells were not included in the spheroid process and other analyses.

Chemotherapeutics treatment

The amount of PBA powder (P20009, Sigma‒Aldrich, Merck KGaA, Darmstadt, Germany) was determined by mass-molarity calculation, mixed with RPMI medium, and then sonicated for 10 minutes at 35°C. After the concentrations were determined according to viability assays, only the IC₅₀ doses for each treatment duration were applied to the samples for further analysis.

Colchicine, which is known to inhibit tubulin polymerization, was included in the study as a positive control chemotherapeutic. Colchicine was purchased in powder form (C9754, Sigma‒Aldrich, Merck KGaA, Darmstadt, Germany) and dissolved in pure DMSO [29,30]. To avoid DMSO toxicity, the determined doses (0.05 μM-0.5 μM) were prepared by diluting the stock at a ratio of 1:1000 with appropriate media. Considering the possible toxicity of DMSO, viability analysis was also performed on cells treated with only 0.1% DMSO. As no toxicity was observed, a colchicine stock solution was formulated and utilized in the experiments. Experimental groups named according to the applied therapeutics, treatment time, and culture methods are listed in table 1.

Viability Analysis

The dose at which therapeutics kill half of the total cell population is known as the IC₅₀ value of the therapeutic agent [31]. To determine the IC₅₀ values of colchicine and PBA, MTT assays were conducted in 2D cultures, and CellTiter-GLO 3D viability analyses were performed in 3D cultures.

MTT analysis

Since the concentrations applied by Bradke et al. did not result in clear IC50 values in our experiments, the concentrations were revised and increased [32]. MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] viability assay PBA (1 mM-20 mM) and colchicine (0.05 μM-0.5 μM) treatment were applied for 24 and 48 hours. After the treatment, the MTT assay was performed as described by Keskin et al. [33]. Absorbance measurements were recorded at 570 nm using a microplate reader. Each dose in the experimental set was performed in six replicates, and each experiment was repeated three times.

CellTiter-Glo® 3D analysis

The CellTiter-Glo assay is a luminescent analysis that measures cellular viability based on the amount of ATP present in cells. PC3 and LNCaP cells were used for 3D cultures, and the assay offers enhanced lytic capacity for use in microtissues. The half-life of the resulting luminescent signal is at least 3 hours, making CellTiter-Glo a preferred choice for viability analysis in 3D cultures [34].

Following the manufacturer's recommendations, CellTiter-Glo reagent was stored at -20°C and thawed slowly at +4°C one day prior to use. Spheroids were brought to room temperature 30 minutes before application. To ensure a 1:1 ratio of reagent to media, spheroids were transferred into 96-well mat white plates in 100 μL of existing media, and then 100 μL of reagent was added to each well. Afterward, samples were pipetted 8 times by using a multipipette. The plate was kept in a dark box on a shaker for 25 minutes at room temperature. The luminescent signal was recorded using a microplate reader following a total of 30 minutes of incubation. The experimental set comprised 6 replicates of each dose, and each experiment was repeated 3 times.

Colony formation assay

To examine the tumorigenic behavior of cancer cells after antimitotic chemotherapy, colony formation analysis was performed [35]. For the colony formation assay, 500 cells were seeded per well in a 6-well plate, and colonies were grown for a total of 10 days with media changes every two days. To prevent the formation of colonies beyond 10 days, PBA and colchicine treatments were applied at the IC₅₀ doses on days 8 and 9, respectively, according to the treatment duration (24 hours or 48 hours). On the 10th day of the colony formation analysis, samples were fixed with 4% paraformaldehyde (PFA) and stained with 2% crystal violet (ab246816) for 5 minutes. After washing with distilled water to remove excess dye, images of the samples were photographed by a mobile camera. Colonies were counted using ImageJ software. The number of colonies of the treated samples was compared with that of the untreated groups for every treatment duration. Each treatment group was seeded in triplicate, and each experiment was repeated 3 times.

Migration Analysis (Scratch Assay)

The scratch assay is a method used to investigate cancer cells' metastatic behavior in 2D culture (143). Migration ability is defined as the ability of cells to acquire mobility and migrate toward the empty space created between them [36]. For the migration model, cells were seeded into 6-well plates (PC3: 500,000 cells, LNCaP: 700,000 cells), and the media was refreshed every two days. On the 5th day, when the cells reached 70-80% confluency, a straight line was created in the center of the plate using a pipette tip. Wells were washed with Ca^+2- and Mg⁺²-containing PBS to remove any debris. To record the closure of the scratch, images were taken at 0 h, 24 h and 48 h intervals depending on the treatment duration. At the end of the incubation period, the migration rate was calculated as the ratio of the remaining area (Tf) to the initial area (T0). Three replicates were performed for each sample in each experimental set. The experiment sets were repeated three times.

Cell Proliferation Assay (Ki-67 Test)

2D and 3D culture samples were prepared as a single cell suspension. 2D cultured cells were briefly harvested and fixed with 4% PFA at +4^oC for 30 mins. Spheroids obtained from 3D cultures were initially treated with 0.2% trypsin and then sonicated to obtain single cell suspension. Single cells fixed with 4% PFA along with 2D cultured cells.

All samples then permeabilized with Perm buffer according to the manufacturer (TrueNuclear Transcription Factor Buffer Set, BioLegend). Cells were incubated with primary antibody at RT for 15 mins, then washed and stained with fluorochrome-conjugated secondary antibody at RT for 15 mins. Cells were then washed and resuspended in FACS buffer.

Acquisition on Symphony A1 (BD Bioscience, USA) is held and FlowJo (BD Bioscience, USA) software is used for analysis.

Immunofluorescence Analysis

For 2D culture samples, all cell lines were seeded onto 12-well chambers (81201, IBIDI GmbH, Germany) and allowed to adhere to the surface overnight. The next day, PBA and colchicine were applied at the determined IC₅₀ doses for 24 and 48 hours. After removing the media from the wells, the samples were fixed with 4% PFA at room temperature for 20 minutes. Wells were washed three times with PBS. Permeabilization was achieved with 0.5% Triton-X solution at room temperature for 10 minutes. Then, the samples were washed three times with PBS. The samples were treated with blocking solution (0.3% Triton-X, 5% goat serum) at room temperature for 1 hour. The primary antibodies anti-JNK1+JNK2+JNK3 (1:200, sc-7345, Santa Cruz Biotechnology, USA), anti-p38 (1:100, 9212, Cell Signaling Technology, USA), and anti-ERK1/ERK2 (1:200, sc-135900, Santa Cruz Biotechnology, USA) were prepared in dilution buffer. After removing the blocking solution, plates were incubated in a humid box at +4°C overnight. After washing with PBS, the plates were incubated with anti-JNK1+JNK2+JNK3 and anti-ERK1/ERK2 (goat anti-mouse 568, 1:1000, Ab175473, Abcam, UK) and anti-p38 (goat anti-rabbit 488, 1:1000, Ab150077, Abcam, UK) secondary antibodies at room temperature in the dark for 1 hour. Nuclei were stained with (4′,6-diamidino-2-phenylindole) DAPI for counterstaining. After removing the IBIDI chambers from the glass slides, the samples were gently closed with Floromount, and images were obtained using a Zeiss LSM 800 microscope (Zeiss, Germany). The whole 3D immunofluorescence protocol was performed in eppendorf tubes with a liquid volume of 100 μl for each step similar to 2D protocol. For permeabilization, samples were treated with a 0.5% Triton-X solution at room temperature for 10 minutes. Thereafter, the primary and secondary antibody incubation times increased. For imaging, the spheroids were transferred to 12-well IBIDI chambers with 30 μL of media. Images were obtained using a Zeiss LSM 800 (Zeiss, Germany) microscope without removing the chamber. Intensity measurements were performed using ImageJ software. The intensity data of 2D culture samples were measured on three different areas with a 10X objective for each group. The intensity data were obtained by dividing the intensity of one cell by the area of every cell in the selected era. For 3D samples, the spheroids were imaged using the Z-stack method on a confocal microscope. After combining all the layers for analysis, the total protein intensity of the spheroid was measured. The intensity value was divided by the measurement area to obtain data specific to the sample. Subsequently, for 2D and 3D intensity analysis, the results were normalized based on the intensity value of the negative control sample.

Transmission Electron Microscopy

Colchicine- and PBA-treated 2D cells were detached from the plate using trypsin and mixed with 5% FBS containing medium to inhibit trypsinization. After centrifugation at 1200 rpm for 5 minutes, the resulting cell pellet was fixed with 2.5% glutaraldehyde. The 3D culture samples were transferred within 30 µL media to eppendorf tubes containing 2.5% glutaraldehyde for fixation. The fixed 2D and 3D samples were then embedded in 1.5% agar and incubated for further processing.

The agar-embedded samples were then washed with a buffer solution, postfixed with 1% osmium tetroxide for an hour, and dehydrated in an ascending series of acetone (30%, 60%, 90%, and 100%) for 30 minutes each. The Epoxy-Embedding Kit (Sigma 45359) protocol was used. The molded samples were left at 60°C for 24 hours to polymerize the epon. Semithin sections of 1 µm were obtained using an ultramicrotome (Leica Ultracut R) from the blocks. The sections were stained with 1% toluidine blue, and the area of interest was determined by light microscopy. The blocks were then trimmed accordingly, and ultrathin sections of 60 nm were obtained and placed on 200 mesh copper grids. Sections with a thickness of 1 μm were obtained from the epoxy blocks, and areas, where 60 nm sections would be taken, were identified and photographed after staining with toluidine blue. Untreated 2D culture model samples were obtained by trypsinization and centrifugation to form pellets, resulting in the cells being scattered and losing their original morphologies due to the disconnection of cell‒cell and cell-surface adhesions. Since individual cells were visible in the 2D samples, it was easy to distinguish whether the cells in the image had a normal or apoptotic appearance. The sections were examined and photographed using a Zeiss Gemini SEM 500.

Statistical Analysis

The absorbance values obtained from the microplate reader in the viability analysis were normalized using the GraphPad Prism program, and the IC₅₀ values were obtained by taking the logarithm of the treated concentrations. For the colony formation analysis, the counted colonies were analyzed statistically using one-way ANOVA, Tukey's posttest, and Dunnett's posttest. In the migration analysis, following the closure ratio calculated via Excel the data were analyzed statistically using one-way ANOVA and Tukey's posttest. For the immunofluorescence analysis, the data were analyzed using one-way ANOVA and Dunnett's posttest in GraphPad Prism software, comparing with the control group and with each other experimental group. The statistical significance value was evaluated as p≤ 0.05.

Viability assays

The graphs of the 2D and 3D culture methods showing the IC₅₀ values for 24 and 48 hours of chemotherapeutics treatments in LNCaP, PC3, and RWPE-1 cells are presented (Fig. 1). When the calculated IC₅₀ value for the chemotherapeutic applied to RWPE-1 cells was compared to the IC₅₀ value for the same chemotherapeutic and time category applied to PC3 and LNCaP cells, it was observed that the values for the L4P, P2P, and P4P groups were lower than those for the R2P and R4P groups. In the viability analysis of 48 hours of colchicine treatment to PC3 and LNCaP cell lines, higher IC₅₀ values were obtained than the applied dose range (Fig. S1). In one of many repeated viability analyses of 24-hour colchicine-treated PC3 and LNCaP spheroids, once an IC₅₀ value in the treated range was obtained and applied for further experiments. The PBA and colchicine IC₅₀ values of PC3 and LNCAP cells after 24 and 48 hours of treatment are summarized in the table at the end of figure 1.

Colony formation assay

The treatment durations of chemotherapeutics were compared with those of the untreated group. Images and graphical analyses of LNCaP colonies treated with PBA and colchicine are presented in figure 2A. The statistical analyses showed a significant decrease (p=0.011) in colony counts of the L2P group compared to the untreated group. On the other hand, a significant decrease (p=0.006) in colony counts was observed in L2P compared to L2C. No statistically significant difference was found in colony counts of the L4P group. The images of PC3 colony petri dishes and graphical analyses are presented in figure 2B. Statistical analyses showed a significant decrease (p=0.038) in the P2P group compared to the untreated group. In addition, a significant decrease (p=0.042) was noted in the P2P group compared to the P2C group. A significant decrease (p=0.022) was observed in P4C compared to the untreated group. There was no statistically significant difference between the 48 hours treated PBA and colchicine groups.

Migration assay

The effects of PBA and colchicine treatment on migration in LNCaP cells were analyzed by scratch test with dependent measurements in the same area (Figure 3A). Statistical analysis revealed that the migration rate was significantly reduced in the L2P (p=0.0002), L2C (p=0.0001), and L4P (p=0.002) groups compared to the untreated group. When the 48 hours

chemotherapeutic treatment groups were compared, it was observed that there was a statistically significant decrease in the L4P group compared to the L4C group (p=0.009).

The effects of PBA and colchicine on migration in PC3 cells were analyzed by scratch test with dependent measurements in the same area (Figure 3B). Statistical analysis revealed that there was a statistically significant decrease in the migration rate in the P2P (p=0.013) and P2C (p=0.015) groups compared to the untreated group. There was no statistically significant difference between the 24 hours PBA and colchicine treatment groups. The migration rate significantly decreased in the P4P (p=0.002) and P4C (p=0.026) groups. There was no statistically significant difference between the 48 hours chemotherapeutics groups.

Proliferation Assay

The flow cytometry graphs for LNCaP and PC3 cells subjected to chemotherapeutic applications at 24 and 48 hours were obtained in the 2D and 3D culture models (Figure S2). The changes in proliferation results were calculated as the percentage of flow cytometry data difference between the PBA treated and untreated groups (Table 2). In the 2D culture model, a decrease in proliferation of 69,68% in the L2P group, 53,51% in the L4F group, 45,90% in the P2P group, and 40,56% in the P4P group was observed. In the 3D culture model, a decrease of 98% in the L2P group, 90,39% in the L4P group, 35,01% in the P2P group, and 13,30% in the P4F group was observed.

Table 2. Proliferation inhibition ratio (%) of PBA treated LNCaP and PC3 cells groups

	L2P	L4P	P2P	P4P
2D	69,68	53,51	45,90	40,56
3D	98,73	90,39	35,01	13,30

Immunofluorescence Analysis

Fluorescence images and intensity analyses graphics of ERK, JNK, and p38 immunostaining were obtained for LNCaP and PC3 cells in 2D and 3D culture models after 24 and 48 hours PBA and colchicine treatments (Figure 4). The immunofluorescence intensity measurements were statistically analyzed by normalization to the negative control samples treated only with the secondary antibody.

In LNCaP cells in the 2D culture model, a statistically significant decrease in the expression of ERK was observed in the L4P (p=0.021) and L4C (p=0.013) groups compared to the untreated group. In the intergroup statistical comparison, there was a decrease in the expression of ERK in the L4P group (p=0.006) compared to the L2P group, in the L4C group (p=0.004) compared to the L2P group, and in the L4C group (p=0.033) compared to the L2C group. In the 3D culture model, a statistically significant decrease in the expression of ERK was observed in the L2P (p=0.048) group compared to the untreated group, and a statistically significant increase in the expression of ERK was observed in the L2C group (p<0.0001). There was a statistically significant decrease in the expression of ERK in the L2P (p<0.0001) and L4P (p<0.0001) groups compared to the L2C group in the intergroup statistical comparison. In the PC3 cells of the 2D culture model, a statistically significant increase in the expression of ERK was observed in the P4C group (p=0.001) compared to the untreated group. In the intergroup statistical comparison, there was a statistically significant decrease in the expression of ERK in the P2P (p=0.04), P2C (p=0.021), and P4P (p=0.004) groups compared to the P4C group.

In the 3D culture model, a statistically significant decrease in the expression of ERK was observed in the P2C (p=0.009) and P4P (p=0.0007) groups compared to the untreated group. In the intergroup statistical comparison, there was a statistically significant decrease in the expression of ERK in the P2C (p=0.01) and P4P (p=0.007) groups compared to the P2P group and in the P4P group (p=0.01) compared to the P2C group.

Transmission Electron Microscopy

In semi thin sections, it was observed that the cells treated with PBA and colchicine either grew in volume or became much smaller due to the apoptotic process, and the apoptotic cells were much more abundant than in the untreated group. In the 3D culture model, all spheroids were taken as samples, and it was observed that the cells maintained their characteristic morphologies without losing their connections with each other. When the PBA and colchicine treated groups were examined, lacunar areas formed by the disconnection of cell‒cell adhesions within spheroid integrity were observed. It was also observed that spheroid sizes were smaller than those in the untreated groups (Figure 5A).

Sections with a thickness of 60 nm obtained from the epoxy blocks were evaluated ultrastructurally. In the 2D and 3D culture models, chemotherapy treatments of LNCaP and PC3 cells resulted in apoptotic lamellar vacuoles, vacuoles, apoptotic blebbing on the cell membrane, and double nucleolus as apoptotic markers (Figure 5B). In the 3D samples, it was observed that the cellular structures used for cellular communication, containing cytoskeletal content, underwent deformation and showed regression in the membrane because the cells were still connected to each other.

The preference for 3D culture models in cancer research is primarily driven by their ability to serve as experimental models that closely resemble the clinical response to chemotherapy [37]. Since cells are arranged in a spherical structure, spheroids are exposed to varying levels of oxygen, nutrients, signaling molecules, and metabolites at different depths [10]. Therefore, differences in drug exposure may lead to distinct responses between 2D and 3D culture models [38]. The knowledge of PBA is based on a very limited background. Our study provides considerable insight into the anticarcinogenic effect of PBA.

Our viability analysis results show that IC₅₀ values obtained from 3D models for each sample were 2–3 times higher than those obtained from 2D models. Our findings suggest that PBA IC₅₀ values obtained from 3D models might be more adaptable to clinical contexts in prospective investigative pursuits. It is noted that the significant disadvantage of antimitotic chemotherapeutics is their potential toxicity [38, 39]. In our study, the toxicity ratio provides insights into the potential toxicity concentration of the chemotherapeutics on RWPE-1 cells. This ratio was calculated by normalizing the IC₅₀ values of LNCaP and PC3 cells with RWPE-1 cells. There are negative results that may suggest no toxicity on normal epithelial cells while that concentration is obviously toxic on cancer cells. Our calculations also reveal a proportional increase in the toxicity ratio of colchicine, while the PBA toxicity ratio decreases over time. From this perspective, PBA might have a significant advantage versus one of the most used chemotherapeutics. The quantitative findings in our study attest to the non-induction of toxicity in healthy epithelial cells by PBA, thereby establishing a foundational benchmark for subsequent investigations into the toxicity profile of PBA. These inquiries evaluate its appropriateness for potential application in clinical interventions for prostate cancer. As a consequence of repeating the viability analysis of both of the 12 and 48 hours colchicine treatment groups, the IC₅₀ values were obtained at higher concentrations than the treated. This observation suggested the possibility of colchicine resistance development in both 12- and 48-hour treatments. There are similar results in the literature reporting colchicine resistance in cells has been described as the ability of cells to avoid the anticarcinogenic effect of the drug due to changes in the signaling mechanisms induced by colchicine [40, 41]. Although colchicine could be used for different diseases, its many side effects, including immune system disorders, require attention [42]. PBA has a clear preference advantage for its inhibitory effect on carcinogenesis due to no resistance in the samples, as evidence of a sustainable metabolic effect over colchicine treatment even in long-term low-dose treatment.

Notably, many studies on prostate cancer treatments aim to halt the metastatic process of the disease [43–46]. In line with the literature, the migration analysis known as the scratch test showed differences in metastatic behaviors between groups. In a study comparing the effects of boric acid and PBA on migration in the castration-independent cell line DU-145, PBA was reported to inhibit migration to a greater extent [19]. Migration was significantly inhibited in all LNCaP cell line groups, except for L4C, which shows the advantage of PBA treatment in a time dependent manner. In PC3 cells, migration was significantly inhibited in all groups. Additionally, both chemotherapeutics showed a higher migration inhibition ratio after 24 hours than after 48 hours of treatment. Colchicine lost its migration inhibition effectiveness over time. In contrast, the migration inhibition rate was consistently over 50% in both the 24- and 48-hour PBA treatment groups. Furhermore, colony formation analysis, PBA has a significant inhibitory effect in both cancer cells. Colchicine inhibits the tumourigenicity of the PC3 cell line when the treatment time is extended to 48 hours. Conversely, PBA effectively inhibited colony formation in both cell lines in short-term high-dose treatment. PBA appears to extend its potent anti-metastatic effect irrespective of treatment duration.

Cancer cells utilize components of JNK signaling pathways in regulating cellular transformation, proliferation, and tumor development processes [47–49]. The decrease in the JNK protein expression ratio in 24-hour PBA-treated PC3 and LNCaP cell lines confirmed our colony and migration analyses, representing the nature of cancer cells, such as tumor development and cellular transformation. Furthermore, in the 3D culture model, a significant decrease in the JNK expression ratio was also observed in the L2P group. Suppressing JNK expression might be one of the anticarcinogenic effects of 24-hour PBA treatment on LNCaP cells as reported the prosurvival function of JNK protein, which includes triggering cancer cell proliferation, invasion and migration [50–53]. In the PC3 cell line groups, a decreased JNK expression ratio was observed only in the 2D P2P group. The lack of an expression ratio difference in the PBA-treated spheroids and 2D P4P groups might suggest that PC3 cells may overcome the suppression of the JNK protein by PBA or that PBA could not sustain the inhibitory effect for 48 hours of treatment or 3D culture models. It has been observed that in both culture models and cells, PBA leads to a slight increase and approaches the untreated groups’s JNK expression ratio in a time-dependent manner. JNK is inhibited in tumor cells due to its tumor suppressive role [47]. The decrease in the expression ratio observed in the 24-hour PBA-treated 2D groups suggests the possible suppression of JNK in the primary response of prostate cancer cells. However, it indicates that after a 48-hour treatment duration, it may have overcome the inhibitory effect. Based on these findings, it may not be a suitable objective to target JNK with PBA treatment in prostate cancer as JNK stabilizes over time. The literature indicates that the JNK protein regulates the cytoskeleton and microtubule dynamics in healthy cells [25]. A study conducted in PC3 and LNCaP cells revealed that decreased JNK activation reduced cell proliferation and migration, ultimately leading to apoptosis [54]. Therefore, the accompanying decrease in the JNK expression ratio in the 24-hour PBA treatment groups, along with colony and migration inhibition, may imply that JNK pathway might be one of the regulator mechanisms of PBA’s effect on microtubule dynamics. Kolomeichuk et al. reported a positive feedback loop between JNK and microtubules [55]. Microtubule-disrupting chemotherapeutic agents target the proliferation process that causes cell cycle arrest in cancer cells [26, 56]. The literature indicates that PBA and boric acid inhibit proliferation in cancer cell lines, including prostate cancer cells [17, 19, 27, 56]. In addition to the known antimitotic effect of colchicine, it may cause an increase in proliferation [57]. It may be suggested that the proliferation increase in colchicine-treated LNCaP and PC3 cells might result from the compensatory effect caused by colchicine. The drug resistance encountered in the 3D culture model against PC3 and LNCaP cells partially supports these data.

Another stress-activated protein, p38, is also included in apoptosis metabolism [58–60]. Moreover, the statistically significant increase in p38 and decrease in the JNK expression ratio observed in L2P spheroids compared to the untreated group may indicate that these cells may activate the p38-mediated apoptotic response under stress. The difference between PBA treatment durations can be observed in monolayer analysis but did not reach the significance level. However, there was a significant p38 expression ratio decrease in PBA-treated LNCaP spheroids in a time-dependent manner. According to these results, it may be suggested that LNCaP cells may exhibit different metabolic responses to PBA treatment in the 2D and 3D models. In PC3 cells, PBA treatment increased the p38 expression ratio in both culture models except for P2P monolayer samples. Therefore, it can be concluded that PBA may have an inductive effect on the p38 kinase in PC3 cells.

Our study demonstrates that PBA treatment inhibits proliferation in prostate cancer cells. When comparing the proliferation analysis between 2D and 3D models, it can be observed that the statistically significant inhibition seen in the 3D model is more apparent in LNCaP cells, while it is relatively lower in PC3 cells. The underlying reason might be the strong metastatic ability, high metabolic activity, and extreme adaptation ability of PC3 cells. It might also be pointed out that PBA sustained its antiproliferative effect on LNCaP spheroids time independent. In correlation with the proliferation analysis, there was a statistically significant decrease in the ERK expression ratio in L2P spheroids. ERK has an essential role in cell proliferation by maintaining proliferation signals and having crosstalk with other important proliferation pathways [54]. The statistically significant increase in the p38 expression ratio in L2P spheroids suggests that p38 might have a role in apoptosis along with ERK-mediated inhibition of proliferation. It is also reported that in cancer ERK has a double effect on proliferation and apoptosis [61].

Both chemotherapeutic treatments in the 2D LNCaP culture samples led to a time-dependent decrease in the ERK expression ratio. When comparing the ERK expression alterations of PBA-treated PC3 cells in both culture models, it was observed that PBA did not affect ERK expression in the 2D model, whereas in the 3D model, there was a decrease in the ERK expression ratio in a time-dependent manner. These two different outcomes indicate that PC3 cells may respond differently to PBA treatment via ERK protein in 2D and 3D models. It may be suggested that decreasing ERK expression in PC3 cells may require long-term treatment. The analysis of the ERK expression ratio showed a statistically significant increase in the L2C spheroid and 2D P4C groups. The same molecular response was shared between different cells and treatment groups, revealing the metabolic differences between the cells. It is also known in the literature that microtubule-disrupting agents such as colchicine can lead to ERK activation [62, 63]. The increase in ERK expression ratios may indicate that colchicine may have triggered some molecular responses, including ERK-mediated apoptosis, migration, or invasion. Similar to Lim et al.’s report, the lack of an increase in the p38 expression ratio in these groups suggests that the cells may carry out their apoptotic responses via ERK [54]. On the other hand, it is noted that the ERK MAPK protein can be activated by multiple stimuli within the cell, as well as by MAPKK [58, 64]. A study conducted on DU-145 cells reported that apoptotic induction increased the level of phosphorylated ERK[27]

This study also examined the morphological changes caused by chemotherapy and changes in colony formation, migration behavior, proliferative attitude, and molecular metabolic responses in cancer cells at the ultrastructural level. The first micrographs of ultrastructural markers indicating cell death, such as the disrupted nucleus and cell membrane, apoptotic vesicle formation, deterioration in filopodia structures, vacuolization, and lamellar vacuole accumulation, were observed in our study as the antineoplastic effects of PBA on prostate cancer cells.

According to the results, antimitotic chemotherapeutic agents may exhibit different metabolic responses in different cells at different doses and durations, depending on whether the cells contain androgen receptors and the differences in the signaling mechanisms and stimuli. Therefore, investigating both castration-dependent and castration-resistant metabolic signaling mechanisms is essential for beneficial prostate cancer therapy research. Our study demonstrates that PBA has an anticancer effect potential including inhibiting proliferation and migration. Additionally, its lower toxicity is a great advantage to take the study to forward deeper investigations. This study presents the first transmission electron microscope images of morphological disorders caused by PBA in human prostate cancer cells. Additionally, as a natural compound, the lower toxicity of PBA on RWPE-1 was reported for the first time. This is a remarkable result for a step forward as a potential chemotherapeutic in future research.

It is needed to analyze more detail on molecular pathways to understand the anticarcinogenic effect of PBA. The limitation of our study is research the limited number of molecules. Further detailed molecular research designs are our next step.

2D: Two dimensional

3D: Three dimensional

LU: Untreated LNCaP cells

LU2: Untreated LNCaP cells (24 hour)

LU4: Untreated LNCaP cells (48 hour)

L1C: 12 hours colchicine treated LNCaP cells

L2C: 24 hours colchicine treated LNCaP cells

L2P: 24 hours phenylboronic acid treated LNCaP cells

L4C: 24 hours colchicine treated LNCaP cells

L4P: 48 hours phenylboronic acid treated LNCaP cells

PU: PC3 Untreated

PU2: PC3 Untreated (24 hours)

PU4: PC3 Untreated (48 hours)

P1C: 12 hours colchicine treated PC3 cells

P2C: 24 hours colchicine PC3 cells

P2P: 24 hours phenylboronic acid treated PC3 cells

P4C: 48 hours colchicine treated PC3 cells

P4P: 48 hours phenylboronic acid treated PC3 cells

PBA: Phenylboronic acid

Ethical approval and consent to participate

This study was ethically approved by Istanbul Medipol University Ethical Committee with E-10840098-772.02-6587 number.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests

Funding

This study is supported by Istanbul Medipol University Scientific Research Project.

Authors' contributions

The study was hypothesized by DGG, MBT and İK. DG performed the cell culture experiments and viability, scratch, colony, and immunofluorescence analysis. DG, AG, and AAK performed transmission electron microscopy analysis. DG and OK performed proliferation analysis. MBT and İK were major contributors in writing the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable

Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69:7–34. https://doi.org/10.3322/caac.21551.
Palmberg C, Koivisto P, Visakorpi T, Tammela TLJ. PSA decline is an independent prognostic marker in hormonally treated prostate cancer. Eur Urol 1999;36:191–6. https://doi.org/10.1159/000067996.
Türk C, Neisius A, Petřík A, Seitz C, Thomas K, Skolarikos A. EAU Guidelines on Urolithiasis 2020. European Association of Urology Guidelines 2020 Edition 2020;presented.
Berish RB, Ali AN, Telmer PG, Ronald JA, Leong HS. Translational models of prostate cancer bone metastasis. Nat Rev Urol 2018;15:403. https://doi.org/10.1038/s41585-018-0020-2.
Ramjan A, Hossain M, Runa JF, Md H, Mahmodul I. Evaluation of thrombolytic potential of three medicinal plants available in Bangladesh, as a potent source of thrombolytic compounds. Avicenna J Phytomed 2014;4:430–6. https://doi.org/10.22038/ajp.2014.3226.
George K, Thomas NS, Malathi R. Modulatory Effect of Selected Dietary Phytochemicals on Delayed Rectifier K + Current in Human Prostate Cancer Cells. Journal of Membrane Biology 2019;252:195–206. https://doi.org/10.1007/s00232-019-00070-9.
Le Page C, Koumakpayi IH, Alam-Fahmy M, Mes-Masson A-M, Saad F. Expression and localisation of Akt-1, Akt-2 and Akt-3 correlate with clinical outcome of prostate cancer patients. Br J Cancer 2006;94:1906–12. https://doi.org/10.1038/sj.bjc.6603184.
Lin RZ, Chang HY. Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnol J 2008;3:1172–84. https://doi.org/10.1002/biot.200700228.
Imamura Y, Mukohara T, Shimono Y, Funakoshi Y, Chayahara N, Toyoda M, et al. Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. Oncol Rep 2015;33:1837–43. https://doi.org/10.3892/or.2015.3767.
Langhans SA. Three-dimensional in vitro cell culture models in drug discovery and drug repositioning. Front Pharmacol 2018;9:6. https://doi.org/10.3389/fphar.2018.00006.
Costa EC, Moreira AF, de Melo-Diogo D, Gaspar VM, Carvalho MP, Correia IJ. 3D tumor spheroids: an overview on the tools and techniques used for their analysis. Biotechnol Adv 2016;34:1427–41. https://doi.org/10.1016/j.biotechadv.2016.11.002.
Riedl A, Schlederer M, Pudelko K, Stadler M, Walter S, Unterleuthner D, et al. Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT-mTOR-S6K signaling and drug responses 2017. https://doi.org/10.1242/jcs.188102.
Penland JG. Dietary Boron, Brain Function, and Cognitive Performance. 1994.
Murphy BT, MacKinnon SL, Yan X, Hammond GB, Vaisberg AJ, Neto CC. Identification of triterpene hydroxycinnamates with in vitro antitumor activity from whole cranberry fruit (Vaccinium macrocarpon). J Agric Food Chem 2003;51:3541–5. https://doi.org/10.1021/jf034114g.
Li X, Wang X, Zhang J, Hanagata N, Wang X, Weng Q, et al. Hollow boron nitride nanospheres as boron reservoir for prostate cancer treatment. Nat Commun 2017. https://doi.org/10.1038/ncomms13936.
Marasovic M, Ivankovic S, Stojkovic R, Djermic D, Galic B, Milos M. In vitro and in vivo antitumour effects of phenylboronic acid against mouse mammary adenocarcinoma 4T1 and squamous carcinoma SCCVII cells. J Enzyme Inhib Med Chem 2017;32:1299–304. https://doi.org/10.1080/14756366.2017.1384823.
Gallardo-Williams MT, Chapin RE, King PE, Moser GJ, Goldsworthy TL, Morrison JP, et al. Boron Supplementation Inhibits the Growth and Local Expression of IGF-1 in Human Prostate Adenocarcinoma (LNCaP) Tumors in Nude Mice. Toxicol Pathol 2004;32:73–8. https://doi.org/10.1080/01926230490260899.
T.Barranco W, Paul FH, Eckhert CD. Evaluation of ecological and in vitro effects of boron on prostate cancer risk (United States). Cancer Causes Control (2007) 2007;18:71–7. https://doi.org/10.1007/s10552-006-0077-8.
McAuley EM, Bradke TA, Plopper GE. Phenylboronic acid is a more potent inhibitor than boric acid of key signaling networks involved in cancer cell migration. Cell Adh Migr 2011;5:5:382–6. https://doi.org/10.4161/cam.5.5.18162.
Psurski M, Łupicka-Słowik A, Adamczyk-Woźniak A, Wietrzyk J, Sporzyński A. Discovering simple phenylboronic acid and benzoxaborole derivatives for experimental oncology – phase cycle-specific inducers of apoptosis in A2780 ovarian cancer cells. Invest New Drugs 2019;37:35–46. https://doi.org/10.1007/s10637-018-0611-z.
Kaur R, Kaur G, Gill RK, Soni R, Bariwal J. Recent developments in tubulin polymerization inhibitors: An overview. Eur J Med Chem 2014;87:89–124. https://doi.org/10.1016/j.ejmech.2014.09.051.
Qin M, Peng S, Liu N, Hu M, He Y, Li G, et al. LG308, a novel synthetic compound with antimicrotubule activity in prostate cancer cells, exerts effective antitumor activity. Journal of Pharmacology and Experimental Therapeutics 2015;355:473–83. https://doi.org/10.1124/jpet.115.225912.
Mukhtara E, Adhamia VM, Sechib M, Mukhtara H. Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells. Cancer Letter 2015;367:173–183. https://doi.org/10.1016/j.physbeh.2017.03.040.
Darcy Bates C, Bates D, Eastman A. Microtubule destabilising agents: far more than just antimitotic anticancer drugs. Br J Clin Pharmacol 2017;83:255–268. https://doi.org/10.1111/bcp.13126.
Stone AA, Chambers TC. Microtubule inhibitors elicit differential effects on MAP kinase (JNK, ERK, and p38) signaling pathways in human KB-3 carcinoma cells. Exp Cell Res 2000;254:110–9. https://doi.org/10.1006/excr.1999.4731.
Shtil AA, Mandlekar S, Yu R, Walter RJ, Hagen K, Tan TH, et al. Differential regulation of mitogen-activated protein kinases by microtubule-binding agents in human breast cancer cells. Oncogene 1999;18:377–84. https://doi.org/10.1038/sj.onc.1202305.
Barranco WT, Eckhert CD. Cellular changes in boric acid-treated DU-145 prostate cancer cells. Br J Cancer 2006;94:884–90. https://doi.org/10.1038/sj.bjc.6603009.
LNCaP Cell Line Spheroid Generation and Characterization for HT Assays. Thermo Fisher Scientific n.d. https://www.thermofisher.com/tr/en/home/references/protocols/cell-culture/3-d-cell-culture-protocol/lncap-cell-line-spheroid-generation.html.
Oh J, An J, Jeong Yeo H, Choi S, Oh J, Kim S, et al. Colchicine as a novel drug for the treatment of osteosarcoma through drug repositioning based on an FDA drug library 2022. https://doi.org/10.3389/fonc.2022.893951.
Kurek J, Myszkowski K, Okulicz-Kozaryn I, Kurant A, Kamińska E, Szulc M, et al. Cytotoxic, analgesic and anti-inflammatory activity of colchicine and its C-10 sulfur containing derivatives n.d. https://doi.org/10.1038/s41598-021-88260-1.
Sebaugh JL. Guidelines for accurate EC50/IC50 estimation. Pharm Stat 2011;10:128–34. https://doi.org/10.1002/PST.426.
Bradke TM, Hall C, Carper SW, Plopper GE. Phenylboronic acid selectively inhibits human prostate and breast cancer cell migration and decreases viability. Cell Adh Migr 2008;2:153–60. https://doi.org/10.4161/cam.2.3.6484.
Bulbul MV, Karabulut S, Kalender M, Keskin I. Effects of Gallic Acid on Endometrial Cancer Cells in Two and Three Dimensional Cell Culture Models. Asian Pacific Journal of Cancer Prevention 2021;22:1745–51. https://doi.org/10.31557/APJCP.2021.22.6.1745.
Farfan A, Yeager T, Moravec R, Niles A. MULTIPLEXING HOMOGENEOUS CELL-BASED ASSAYS. CELL NOTES 2004:15–8.
Yang H, Ganguly A, Cabral F. Inhibition of Cell Migration and Cell Division Correlates with Distinct Effects of Microtubule Inhibiting Drugs. J Biol Chem 2010;285:32242. https://doi.org/10.1074/JBC.M110.160820.
Martinotti S, Ranzato E. Scratch Wound Healing Assay. Methods Mol Biol 2020;2109:225–9. https://doi.org/10.1007/7651_2019_259.
Fontana F, Raimondi M, Marzagalli M, Sommariva M, Gagliano N, Limonta P. Three-dimensional cell cultures as an in vitro tool for prostate cancer modeling and drug discovery. Int J Mol Sci 2020;21:1–18. https://doi.org/10.3390/ijms21186806.
Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007;2:329–33. https://doi.org/10.1038/NPROT.2007.30.
Huang H, Li LJ, Zhang HB, Wei AY. Papaverine selectively inhibits human prostate cancer cell (PC-3) growth by inducing mitochondrial mediated apoptosis, cell cycle arrest and downregulation of NF-κB/PI3K/Akt signalling pathway. Journal of BUON 2017;22:112–8.
Kumar A, Sharma PR, Mondhe DM. Potential anticancer role of colchicine-based derivatives: an overview. Anticancer Drugs 2017;28:250–62. https://doi.org/10.1097/CAD.0000000000000464.
Kaushik V, Yakisich JS, Way LF, Azad N, Iyer AKV. Chemoresistance of cancer floating cells is independent of their ability to form 3D structures: Implications for anticancer drug screening. J Cell Physiol 2019;234:4445–53. https://doi.org/10.1002/jcp.27239.
Leung YY, Yao Hui LL, Kraus VB. Colchicine-Update on mechanisms of action and therapeutic uses. Semin Arthritis Rheum 2015;45:341–50. https://doi.org/10.1016/j.semarthrit.2015.06.013.
Veine DM, Yao H, Stafford DR, Fay KS, Livant DL. A D-amino acid containing peptide as a potent, noncovalent inhibitor of a5b1 integrin in human prostate cancer invasion and lung colonization n.d. https://doi.org/10.1007/s10585-013-9634-1.
Abel SDA, Dadhwal S, Gamble AB, Baird SK. Honey reduces the metastatic characteristics of prostate cancer cell lines by promoting a loss of adhesion. PeerJ 2018;2018. https://doi.org/10.7717/peerj.5115.
Schatten H. Brief overview of prostate cancer statistics, grading, diagnosis and treatment strategies. Adv Exp Med Biol 2018;1095:1–14. https://doi.org/10.1007/978-3-319-95693-0_1.
Dehghani M, Kianpour S, Zangeneh A, Mostafavi-Pour Z. CXCL12 Modulates Prostate Cancer Cell Adhesion by Altering the Levels or Activities of í µí»½1-Containing Integrins 2014. https://doi.org/10.1155/2014/981750.
Kennedy NJ, Davis RJ. Role of JNK in tumor development. Cell Cycle 2003;2:199–201. https://doi.org/10.4161/cc.2.3.388.
Weston CR, Davis RJ. The JNK signal transduction pathway. Curr Opin Cell Biol 2007;19:142–9. https://doi.org/10.1016/j.ceb.2007.02.001.
Eferl R, Ricci R, Kenner L, Zenz R, David J-P, Rath M, et al. Liver Tumor Development: c-Jun Antagonizes the Proapoptotic Activity of p53 specific functions of c-Jun for cell survival and cell-cycle progression imply that this gene might be a major regulator in the development of hepatic carcinomas. Cell 2003;112:181–92.
He Y, Duckett D, Chen W, Ling YY, Cameron MD, Lin L, et al. Synthesis and SAR of novel isoxazoles as potent c-jun N-terminal kinase (JNK) Inhibitors. Bioorg Med Chem Lett 2014;24:161. https://doi.org/10.1016/J.BMCL.2013.11.052.
Zhao HF, Wang J, Jiang HR, Chen ZP, To SST. PI3K p110β isoform synergizes with JNK in the regulation of glioblastoma cell proliferation and migration through Akt and FAK inhibition. J Exp Clin Cancer Res 2016;35. https://doi.org/10.1186/S13046-016-0356-5.
Zhou Y, Wang Y, Zhao Z, Wang Y, Zhang N, Zhang H, et al. 37LRP induces invasion in hypoxic lung adenocarcinoma cancer cells A549 through the JNK/ERK/c-Jun signaling cascade. Tumour Biol 2017;39. https://doi.org/10.1177/1010428317701655.
Han J, Jeon M, Shin I, Kim S. Elevated STC-1 augments the invasiveness of triplenegative breast cancer cells through activation of the JNK/cJun signaling pathway. Oncol Rep 2016;36:1764–71. https://doi.org/10.3892/OR.2016.4977.
Lim W, Jeong M, Bazer FW, Song G. Coumestrol Inhibits Proliferation and Migration of Prostate Cancer Cells by Regulating AKT, ERK1/2, and JNK MAPK Cell Signaling Cascades. J Cell Physiol 2017;232:862–71. https://doi.org/10.1002/JCP.25494.
Kolomeichuk SN, Terrano DT, Lyle CS, Sabapathy K, Chambers TC. Distinct signaling pathways of microtubule inhibitors–vinblastine and Taxol induce JNK-dependent cell death but through AP-1-dependent and AP-1-independent mechanisms, respectively. FEBS J 2008;275:1889–99. https://doi.org/10.1111/J.1742-4658.2008.06349.X.
Zhou J, Giannakakou P. Targeting microtubules for cancer chemotherapy. Curr Med Chem Anticancer Agents 2005;5:65–71. https://doi.org/10.2174/1568011053352569.
Kamath A, Mehal W, Jain D. Colchicine-associated Ring Mitosis in Liver Biopsy and Their Clinical Implications. J Clin Gastroenterol 2008;42. https://doi.org/10.1097/MCG.0b013e31803815b4.
Kyriakis JM, Avruch J. Mammalian Mitogen-Activated Protein Kinase Signal Transduction Pathways Activated by Stress and Inflammation. 2001.
John M. Kyriakis, Joseph Avruch. Protein kinase cascades activated by stress and inflammatory cytokines. BioEssays 1996;18:567–77.
Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science (1979) 1995;270:1326–31. https://doi.org/10.1126/science.270.5240.1326.
Sugiura R, Satoh R, Takasaki T. ERK: A Double-Edged Sword in Cancer. ERK-Dependent Apoptosis as a Potential Therapeutic Strategy for Cancer. Cells 2021;10. https://doi.org/10.3390/CELLS10102509.
Schmid-Alliana A, Menou L, Manié S, Schmid-Antomarchi H, Millet M-A, Giuriato S, et al. Microtubule Integrity Regulates Src-like and Extracellular Signal-regulated Kinase Activities in Human Pro-monocytic Cells IMPORTANCE FOR INTERLEUKIN-1 PRODUCTION*. Journal of Biological Chemistry 1998;273:3394–400. https://doi.org/10.1074/jbc.273.6.3394.
Nair RR, Schwarz. LA. Microtubule-disrupting Agents Increase Transgene Expression in A549 Cells Through The Activation of The Src and ERK Kinase Pathway. Molecular Therapy 2003;7.
Samarakoon R, Higgins PJ. MEK/ERK pathway mediates cell-shape-dependent plasminogen activator inhibitor type 1 gene expression upon drug-induced disruption of the microfilament and microtubule networks. J Cell Sci 2002;115:3093–103. https://doi.org/10.1242/JCS.115.15.3093.

Table 1 is available in the Supplementary Files section.

No competing interests reported.

FigureS1.48h12hViability.tif
FigureS2.Proliferation.tif
Table1.Groupnames.tif
Table 1. Experimental groups

Download PDF

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

Investigation of the Effect of Phenylboronic Acid on Androgen-Dependent (LNCaP) and Androgen-Independent (PC3) Prostate Cancer Cells via MAP Kinases by 2D and 3D Culture Methods

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

Cell culture

2D cell culture

3D cell culture

Viability Analysis

MTT analysis

CellTiter-Glo® 3D analysis

Colony formation assay

Migration Analysis (Scratch Assay)

Cell Proliferation Assay (Ki-67 Test)

Immunofluorescence Analysis

Transmission Electron Microscopy

Statistical Analysis

RESULTS

Viability assays

Colony formation assay

Migration assay

Proliferation Assay

Immunofluorescence Analysis

Transmission Electron Microscopy

Discussion

Conclusion

Abbreviations

Declarations

References

Table

Additional Declarations

Supplementary Files

Status:

Version 1