Research ArticleExperimental Studies

LQB-118 Suppresses Migration and Invasion of Prostate Cancer Cells by Modulating the Akt/GSK3β Pathway and MMP-9/Reck Gene Expression

THIAGO MARTINO, GRAZIELE FREITAS DE BEM, SHIRLEY VANIA MOURA SANTOS, MARSEN GARCIA PINTO COELHO, ANGELA DE CASTRO RESENDE, CHAQUIP NETTO, PAULO ROBERTO RIBEIRO COSTA, GRAÇA JUSTO and KATIA COSTA DE CARVALHO SABINO
Anticancer Research January 2023, 43 (1) 359-367; DOI: https://doi.org/10.21873/anticanres.16171

Abstract

Background/Aim: Prostate cancer (PCa) is one of the most common malignancies in adult men. LQB-118 is a pterocarpanquinone with antitumor activity toward prostate cancer cells. It inhibits cell proliferation by down-regulating cyclins D1 and B1 and up-regulating p21. However, the effects of LQB-118 on PCa cell migration are still unclear. Herein, the LQB-118 effects on PCa metastatic cell migration/invasion and its mechanism of action were evaluated. Materials and Methods: PC3 cells were treated with LQB-118 or Paclitaxel (PTX), and cell migration (wound healing and Boyden chamber assays) and invasion (matrigel assay) were determined. The LQB-118 mechanisms were evaluated by αVβIII protein expression (flow cytometry), protein phosphorylation (Western blot), and mRNA expression (qPCR). Results: LQB-118 impaired PCa cell migration and invasion, down-regulated Akt phosphorylation, and also reduced GSK3β phosphorylation, through a FAK-independent pathway. Also, it was observed that LQB-118 controlled the invasiveness behavior by reducing matrix metalloproteinase-9 (MMP-9) and up-regulating reversion-inducing cysteine rich protein with Kazal motifs (Reck) mRNA levels. Interestingly, LQB-118 increased integrin αvβIII expression, but this effect was not related to its activation, since the cell adhesion ability was reduced after LQB-118 treatment. Conclusion: These data highlight novel LQB-118 mechanisms in prostate cancer cells. LQB-118 acts as a negative regulator of the Akt/GSK3 signaling pathway and can modulate PCa cell proliferation, death, and migration/invasion. The results also support the use of LQB-118 for the treatment of metastatic PCa, alone or combined with another chemotherapeutic agent, due to its demonstrated pleiotropic activities.

Key Words:

Prostate cancer (PCa) is one of the most prevalent solid tumors and a leading cause of cancer-related deaths among men in the world (1) It is a disease of extensive metastasis, with secondary lesions in the lymph node, brain, bones, and other organs such as the liver and lungs (2). In general, cancer metastasis is a multistep and complex process that involves dissociating the tumor cells from the organ of origin, degradation of the extracellular matrix, cell migration, anchorage-independent growth, angiogenesis, invasion, and colonization to distant sites in the body (3).

Studies have indicated constitutive activation of the PI3K/Akt pathway in advanced PCa since the tumor suppressor PTEN is commonly lost (4, 5). The PI3K/Akt signaling pathway regulates cellular metabolism, tumor development and growth, cytoskeletal reorganization, and metastasis (6). Moreover, Akt has been shown to play an anti-apoptotic role and regulates the activity of glycogen synthase kinase 3-beta (GSK3β) via phosphorylation of the serine-9 residue, resulting in its inactivation (7). Consequently, GSK3β inactivation contributes to cell proliferation, migration, and invasion (8). Although the Akt pathway is extremely important in PCa development, in metastasis the cell migration also depends on proteolytic enzymes, such as matrix metallopeptidase (MMPs), that destroy the extracellular matrix components (ECM) and thereby allow cancer cells to reach other sites (9). Therefore, an important potential approach in cancer therapy may be provided by strategies such as chemotherapy, using agents that are able to prevent, halt, or delay processes including angiogenesis, or modulate the signaling cascades related to cell migration and/or cell proliferation.

A pterocarpanquinone, known as LQB-118, has demonstrated cytotoxic effects on PCa cancer cells, inducing significant cell cycle arrest by down-regulating c-Myc, cyclins D1 and B1 expression, while up-regulating p21 expression (10). Lately, we have correlated LQB-118 activity with quinone reduction, increased NQO1 expression, ROS generation, and apoptosis as a final consequence (11). Furthermore, transient knock-down of SOD1 through siRNA or miRNA caused an increase in PCa cell sensitivity to LQB-118 effects. In vivo efficacy and safety of the LQB-118 antitumor effects were also demonstrated by oral treatment in athymic nude mice bearing PCa tumors (11). However, despite these recent studies on LQB-118, its effects on cell migration and invasion still need to be determined.

Herein, we report on certain new mechanisms of LQB-118 activity related to the inhibition of migratory/invasive PCa cells, including modulation of the Akt/GSK3β signaling pathway, integrin-FAK signaling pathway and expression levels of migration/invasion regulatory genes, such as MMP9 and Reck, in PCa metastatic cells.

Materials and Methods

Cell culture. The androgen-resistant human PCa cell line (PC3) was donated by the Basic Research Department of Rio de Janeiro National Institute Cancer, RJ, Brazil (INCa-RJ). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Cultilab, São Paulo, Brazil), penicillin 70 mg/l, and streptomycin 100 mg/l. Cells were maintained at 37°C in a 5% CO2 humidified atmosphere. The cell lineage was authenticated by the BCRJ (Rio de Janeiro, Brazil) and confirmed to be mycoplasma free.

Chemicals and reagents. The pterocarpanquinone, LQB-118, was synthesized in the Bioorganic Chemistry Laboratory, IPPN, Rio de Janeiro Federal University (Rio de Janeiro, Brazil), as described elsewhere (10), and was kindly donated by Dr. Chaquip Daher Netto and Dr. Paulo Roberto Ribeiro Costa. The maximal DMSO concentration in culture was 0.1% without cytotoxic effects. The matrigel solution was purchased from Sigma Chemical Co., St. Louis, MO, USA, and anti-human pFAK (Y576, ref. 700013), pAkt (pS473, ref. 700392), pGSK3β (pS9, ref. 701069), anti-human GAPDH (ref A21994), and protease inhibitors were purchased from Invitrogen Brasil Ltda, São Paulo, Brazil.

MTT assay. After adhesion in 96 flat-bottom-well microplates, PC3 cells (1×105 cells/ml) were incubated in the absence (control) or presence of different concentrations of LQB-118 at 37°C in a 5% CO2 humidified atmosphere. Cell survival/cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay (Sigma Chemical Co., St. Louis, MO, USA, ref. M2128) after 12 h by incubation at 37°C with 10 μl/well (last 2 h of culture) of MTT stock solution (5 mg/ml) (12). Afterwards, 100 μl/well of SDS 10% solution containing 0.01 N HCl was added to the wells, and the absorbance was measured at 570 nm (microplate reader μQuant, Bio-Tek Instruments, Inc., Winooski, VT, USA). The results are expressed as relative cell viability, using the control culture (without drug) as 100%.

Wound healing migration assay. Cells (1×106/ml) were plated in twenty-four-well culture plates. After cells reached 70% to 80% confluence, PBS (phosphate buffered saline pH 7.4) was added to remove floating cells. Next, all groups were incubated for 24 h with 5 μg/ml Mitomycin C (Sigma Chemical Co., St. Louis, MO, USA, ref. M4287) to avoid cell proliferation (13). Then a sterile pipette tip (10 μl) was used to scratch a line in the middle of the well. Subsequently, the cells were washed twice, and RPMI-1640 serum-free culture medium was replaced. Different images of the denuded area were taken, and at least three random fields per well were captured by light microscopy at 0 h (AnalySIS getIT 5.2, Olympus, USA). PCa cells were then treated with different concentrations of either LQB-118 or Paclitaxel (PTX) for another 12 h, and images were captured again. The qualitative evaluation of cells that migrated after 12 h of incubation in each treatment was compared with that exhibiting in the wound healing produced at time 0 and in relation to the control.

Boyden chamber assay. Cells (2×104/ml) were treated with LQB-118 (1, 3 and 5 μM) or PTX (1 μM) for 12 h. Afterwards, viable cells (2×104/chamber) were washed with PBS, resuspended in a serum-free medium, and placed in the upper compartment. RPMI 1640 medium supplemented with 10% FBS was used as a chemoattractant agent in the lower chamber. The two compartments of the Boyden chamber were separated by 8 μm pore-size polycarbonate filters. After 3 h of incubation at 37°C in 5% CO2, non-migrating cells on the upper chamber were removed and the filters were recovered. They were washed in PBS buffer, fixed in 4% paraformaldehyde for 2 min and permeabilized with 100% methanol for 20 min in room temperature. The filters were rewashed in PBS and then stained with a panoptic dye kit (Laborclin, Paraná, Brazil). The migrated cells were quantified by counting five fields for each insert membrane under a microscope (14).

Invasion assay. Cells (2×104/ml) were treated with LQB-118 (1, 3 and 5 μM) or PTX (1 μM) for 12 h. Then, viable cells (2×104/chamber) were washed with PBS, resuspended in a serum-free medium, and placed in the upper compartment. RPMI 1640 medium supplemented with 10% FBS was used as a chemoattractant agent in the lower chamber. The two compartments of the Boyden chamber were separated by an 8 μm transwell insert which was covered with 60 μg/100 μl of the matrigel, simulating a basement membrane. After 3 h of incubation at 37°C in 5% CO2, the procedure was performed as described above (14).

Flow cytometry. Cells (2×104/ml) were treated either with LQB-118 (1, 3 and 5 μM) or PTX (1 μM) for 12 h at 37°C in 5% CO2. Afterwards, cells were harvested, washed with PBS, and incubated with a mouse anti-human αvβIII integrin – IgG1 (anti-CD51/61) antibody conjugated with Alexa fluor 647 (Biolegend, San Diego, CA, USA, ref. 304410/100) for 30 min at 4°C. Cells were analyzed using a Gallios flow cytometer and Summit software v4.3, Colorado Inc., USA.

RNA isolation and reverse transcription (RT)-quantitative PCR (qPCR). RNA was isolated using TRIzol (Invitrogen). Complementary DNA (cDNA) synthesis was carried out using the Moloney Murine Leukemia Virus Reverse Transcriptase enzyme (M-MLV RT), and qPCR reactions were performed with Fast SYBR Green Master Mix (Life Technologies, São Paulo, Brazil, ref. 4385612), following the manufacturer’s protocols. Alterations in mRNA levels were calculated by the 2−ΔΔCt method (15, 16). All data were normalized using GAPDH as a reference gene. The primer sequences were as following: MMP-9 5′-TGACAGCGACAAGAAGTG-3′ and 5′-CAGTGAAGCGGTACATAG-3′ and Reck 5′-CCTGCATTGCTCGCTGTGTG-3′ and 5′-CCTGTGGTTTGGGTATGCACCTT-3′.

Western blot analysis. Cells were treated as previously described above. Cellular lysates were prepared using RIPA buffer with protease and phosphatase inhibitors. Protein samples were quantified using BSA, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto the nitrocellulose membrane. Membranes were blocked with 5% bovine serum albumin (Sigma, Brazil) for 1 h at room temperature and incubated overnight at 4°C with mouse anti-human antibodies against pFAK (Y576, ref. 700013), pAkt (pS473, ref. 700392) and pGSK3β (pS9, ref. 701069) (1:1,000). After washing, the blots were incubated with peroxidase-conjugated goat anti-mouse secondary antibody (1:10,000) for 1 h at room temperature. The blots were visualized by enhanced chemiluminescence using the ChemiDoc Imaging system (Bio-Rad, São Paulo, Brazil). Signals for each protein were normalized to GAPDH.

Statistical analysis. The results are reported as mean±standard deviation (SD) of at least three independent experiments. Differences among multiple groups were analyzed with one-way analysis of variance (ANOVA) followed by Dunnett’s test. p≤0.05 was indicated as statistically significant. All statistical analysis was performed using GraphPad Prism 7.0 software Inc., La Jolla, CA, USA.

Results

LQB-118 decreases PC3 cell migration at non-cytotoxic concentrations. First, we examined the effects of LQB-118 on PC3 cell migration using a range of concentrations (1 to 10 μM) through a wound healing assay. LQB-118 reduced PCa cell migration since the scratch width did not decrease after 12 h of LQB-118 treatment (Figure 1A). Furthermore, Paclitaxel (PTX), a positive control, also decreased cell migration. Nonetheless, excluding the possibility that LQB-118 decreases cell migration because it inhibits cell proliferation, PC3 cells were previously treated with Mitomycin C (5 μM), which did not show any cytotoxic effect on these cells (13) (data not shown). In parallel, the cytotoxic effects of LQB-118 were also performed under the same conditions used for the wound healing assay. All LQB-118 concentrations evaluated showed no cytotoxic effects compared to the control group (Figure 1B).

Figure 1.
Figure 1.

Effects of LQB-118 on the cell migration and cytotoxicity of androgen-independent prostate cancer (PC3) cells. (A) Representative images of the wound healing analysis of PC3 cells at 0 h and 12 h for cells treated with 1, 5 and 10 μM of LQB-118 or 1 μM of PTX and control cells. (B) Cytotoxic effects of LQB-118 by the MTT assay. LQB-118 effects were assessed as described in Materials and Methods. Results represent the mean±SD of five experiments. *p<0.05 vs. control (untreated cells) by one-way ANOVA followed by Dunnett’s test.

The Boyden chamber assay was also performed using PC3 cells to quantitatively investigate the effects of LQB-118 in cell migration. The percentage of migrated cells after LQB-118 treatment was significantly decreased at 3 and 5 μM (66.7% and 89.3%, respectively) compared to the control culture (Figure 2A). Also, PTX showed a similar effect, inhibiting cell migration at 83.5% at 1 μM (Figure 2A).

Figure 2.
Figure 2.

Inhibition of androgen-independent prostate cancer (PC3) cell migration and invasion by short exposure to LQB-118. (A) PC3 cell migration towards a higher FBS percentage gradient, using a Boyden chamber of 8 μm. (B) PC3 cell invasion through matrigel following an FBS gradient. Cells were previously treated with LQB-118 in different concentrations. Viable cells (2×104) were then placed in the upper compartment in a serum-free medium and migrating and invading cells were analyzed. Results represent the mean±SD of three independent experiments. *p<0.05, **p<0.01 vs. control (untreated cells) analyzed by one-way ANOVA followed by Dunnett’s test.

To explore the possibility that LQB-118 somehow interferes with the PC3 cells’ mobility by decreasing cell migration, we further evaluated whether the effects of LQB-118 also decrease cell invasion. Untreated PC3 cells migrated and invaded through matrigel in response to the FBS-supplemented medium gradient. However, short exposure to 3 μM and 5 μM of LQB-118 significantly decreased PC3 cell invasion by 52.7% and 59%, respectively (Figure 2B). Furthermore, treatment with PTX decreased PC3 cell invasion by 46.8% through matrigel (Figure 2B). Those results might represent a novel mechanism of action of LQB-118 since the cell migration and invasion were decreased at non-cytotoxic concentrations.

Inhibition of PCa cell migration and invasion by LQB-118 is related to reduced MMP-9 and increased Reck gene expression. Therefore, we were next sought to explore the mechanism of action by which LQB-118 reduces PCa cell migration and invasion. Since the metastatic cascade consists of the degradation of ECM by proteolytic enzymes such as MMP-9, we determined the MMP-9 gene expression after LQB-118 treatment. Interestingly, LQB-118 treatment at 3 μM significantly reduced MMP-9 mRNA levels by 33%, while LQB-118 at 5 μM showed a greater inhibition of mRNA levels of 47% (Figure 3A). Likewise, PTX decreased the MMP-9 mRNA levels by 51.1% (Figure 3A) compared to the control culture. To corroborate our findings so far, we also measured the gene expression of a matrix metalloprotease inhibitor, Reck, on PC3 cells after 12 h of treatment. LQB-118 tended to increase Reck mRNA levels at 1 μM (48%) and 3 μM (42.4%). However, at 5 μM, the Reck mRNA expression increased by 86.2% (p<0.001), as well as PTX treatment (120%) at 1 μM (Figure 3B), compared to the control culture.

Figure 3.
Figure 3.

Effects of LQB-118 on matrix metalloproteinase-9 (MMP9) and Reck gene expression in androgen-independent prostate cancer (PC3) cells. (A) MMP-9 mRNA levels. (B) Reck mRNA levels. Cells were incubated either in the absence or presence of LQB-118 (1, 3 and 5 μM) or PTX (1 μM) for 12 h. mRNA levels were evaluated by qPCR. Results represent the mean±SD of three independent experiments. **p<0.01 and ***p<0.001 vs. control (untreated cells) analyzed by one-way ANOVA, followed by Dunnett’s test.

LQB-118 modulates the protein levels of integrin αvβIII at the cell membrane and cell adhesion. To further investigate the LQB-118 effects on PCa cell migration and invasion, we decided to evaluate whether LQB-118 modulates the expression of integrin αvβIII in PC3 cells. To address this question, PC3 cells were treated with different concentrations of LQB-118 (1, 3 and 5 μM) for 12 h, and integrin αvβIII expression was detected by flow cytometry. Surprisingly, LQB-118 treatment increased αvβIII expression in PC3 cells (Figure 4A-C and E), and the positive control PTX (Figure 4D-E). The highest LQB-118 concentration assessed (5 μM) increased integrin expression 1.86-fold, while PTX treatment increased its expression by 3.4-fold (Figure 4E), compared to the untreated culture. As it is known that PCa cell adhesion to components present in the bone matrix is mediated, in part, by αvβIII, we used an adhesion assay to evaluate whether the augmented integrin expression is related to its activity. Even with augmented expression of integrin, LQB-118 treatment, after 12 h, reduced cell adhesion to the surface by 31.9% and 75.2%, respectively (Figure 4F). Likewise, PTX showed a similar effect on PC3 cell adhesion, avoiding 67.6% of cell adhesion to the surface, compared to the control group (Figure 4F).

Figure 4.
Figure 4.

Effects of LQB-118 in αvβIII integrin expression on androgen-independent prostate cancer (PC3) cells after LQB-118 treatment, quantified by flow cytometry. Histogram of control (dashed), treated with LQB-118 (gray) or Paclitaxel (black). (A) Control vs. 1 μM LQB-118, (B) Control vs. 3 μM LQB-118 and (C) Control vs. 5 μM LQB-118. (D) Control vs. 1 μM PTX. (E) Mean fluorescence intensity (MIF) of anti-αvβIII. (F) Percentage of cell adhesion after LQB-118 treatment. Cells were treated with LQB-118 (1, 3 and 5 μM) or PTX (1 μM) for 12 h and integrin expression was measured by flow cytometry and cell adhesion using the MTT assay. Results represent the mean±SD of three independent experiments. **p<0.01 and ***p<0.001 vs. control (untreated cells), analyzed by one-way ANOVA followed by Dunnett’s test.

LQB-118 decrease cell migration and invasion by Akt and GSK3β phosphorylation inhibition. Integrins αvβIII functions are generally mediated by FAK, a non-receptor tyrosine kinase, which activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and is a key pathway for cell proliferation, survival, migration, and invasion. In addition to the augmented αvβIII integrin expression after LQB-118 treatment, short exposure to LQB-118 induced phosphorylation of FAK at Y576 in PC3 cells, regardless of concentrations evaluated, increasing 3.65-fold at 5 μM (Figure 5A, B), when compared to the untreated culture. However, short exposure to LQB-118 significantly decreased phosphorylation of Akt (S473) in PC3 cells, by 23.1%, 50.1% and 61.5% at 1 μM, 3 μM and 5 μM, respectively (Figure 5C, D). Thus, we evaluated the pGSK3β expression, which is phosphorylated at serine 9 by phosphorylated Akt, and we observed a suppressed activity of this Akt downstream target.

Figure 5.
Figure 5.

Effects of LQB-118 on protein expression related to the migration pathway in androgen-independent prostate cancer (PC3) cells. (A) Western blot analysis of pFAK; (B) Mean of FAK phosphorylation at Y576; (C) Western blot analysis of pAkt; (D) Mean of Akt phosphorylation at Ser473, (E) Western blot analysis of pGSK3β; (F) Mean of GSK3β phosphorylation at Ser9. PC3 cells were treated with or without LQB-118 for 4 h, proteins from the cells in each culture condition were extracted, loaded, and separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Results represent the mean±SD of four independent experiments. *p<0.05 and **p<0.01 vs. control (untreated cells) analyzed by one-way ANOVA, followed by Dunnett’s test.

Indeed, since LQB-118 decreased the phosphorylation of Akt, GSK3β phosphorylation was also decreased by LQB-118 at 3 μM and 5 μM (2-fold and 1.65-fold, respectively) (Figure 5E, F).

Discussion

There is a continuous search for new therapeutic agents for advanced and metastatic PCa. Here we examined the effects of the LQB-118 on the migration and invasion of a highly invasive prostate cancer cell line, PC3, and elucidated some mechanisms of its action. It is known that advanced stages of prostate cancer are characterized by a higher risk of developing metastatic disease (2), and emerging evidence indicates that the Akt pathway is constitutively activated in PCa cells, contributing to cell proliferation, survival, inhibition of apoptosis, cell migration and invasion (16, 17).

In addition to the LQB-118 anti-proliferative effect on PC3 cells, previously demonstrated elsewhere (10), we showed that it inhibits the metastatic PCa cell migration through wound healing (Figure 1A) and Boyden chamber assays (Figure 2A), while also inhibiting their invasion property, reducing the number of cells permeating through the matrigel barrier (Figure 2B). Some studies have demonstrated that quinones and derivatives, such as thymoquinone (18), anthraquinone (19), and benzoquinone (20), show cytotoxic effects towards a broad spectrum of cancer and down-regulate cell migration and invasion. Shikonin, a quinone, presents antitumor action related to decreased cell adhesion and inhibition of lung cancer cell migration (21), as observed with LQB-118. Otherwise, treatment with the pterocarpanquinone LQB-118 showed an increase in αvβ3 integrin expression in PC3 cells (Figure 4), as observed with treatment of lung cancer cells with Shikonin. It was suggested that this increase was not associated with Shikonin inhibition of lung cancer cell migration/adhesion (22, 23). Additionally, it has been described that cell anchorage to ECM throughout αvβ3 is critical for driving survival signals, whereas the unbound integrins lead to apoptosis (24, 25).

Adhesion to the ECM precedes the cytoskeleton rearrangement and the transcription of genes, which regulates the fate of tumor cells. Activating these intracellular signaling cascades needs the clustering of αvβ3 on FAK (26). The phosphorylation of FAK in several sites is related to multiple functions, such as cell anchoring, proliferation, and apoptosis in several cell types (27). After a short exposure to LQB-118, PC3 cells augmented αvβ3 integrin expression (Figure 3) and FAK phosphorylation at Y576 (Figure 5A, B). Sonoda and collaborators (28) demonstrated that FAK retained tyrosine phosphorylation by oxidative stress prior to apoptosis, at least up to 5 h, and gradually lost phosphorylation concomitant with apoptosis. Previous study demonstrated oxidative stress in PCa cells treated for 12 h with LQB-118, due to increased NQO1 activity and SOD1 augmented expression (11). Thus, we can suggest that FAK phosphorylation (Y576), mediated by LQB-118, may be induced by oxidative stress and/or by increased αvβ3 expression.

FAK-Src phosphorylation activates PI3K, which generates stimuli for cell proliferation/survival and motility through Akt phosphorylation and Erk kinase transcription (29, 30). Short exposure to LQB-118 rapidly decreased Akt phosphorylation in PC3 cells (Figure 5C, D). Since Akt has a wide spectrum of cellular functions and is also highly activated in prostate cancer, inhibition of Akt phosphorylation is considered a plausible therapeutic approach. Next, we evaluated GSK3β phosphorylation at S9, an Akt-specific downstream phosphorylation site, to corroborate our findings. It is noteworthy that LQB-118 also reduced GSK3β phosphorylation (Figure 5E, F), probably due to the inhibition of Akt phosphorylation (Figure 5C, D).

The constitutive Akt activation in prostate cancer results in GSK3β phosphorylation at S9, switching it to an inactive form (31), thus preventing the phosphorylation of β-catenin and its degradation by the ubiquitin-proteasome pathway (8). In this sense, high levels of β-catenin accumulate and activate target genes promoting proliferation and metastatic behavior (8). Taken together, the LQB-118 results suggest its effects are related to reduced Akt phosphorylation and, consequently, reduced phosphorylation of GSK3β in S9, which turns it active by a FAK-independent pathway. In this case, the level of phosphorylated β-catenin by GSK3 increases, targeting it to proteolysis and inhibition of the cell cycle, previously demonstrated elsewhere (10), as well as metastasis.

A study has demonstrated that the activity of docetaxel on focal adhesion signaling reflects its activity as a microtubule-targeting agent, as microtubules interact with focal adhesion and regulate their turnover (32). Of note, LQB-118 shows a different activity mechanism than docetaxel, which does not involve microtubule targeting.

In addition, we demonstrated that LQB-118 reduced the invasiveness behavior of PC3 cells, and this effect was strengthened by the decreased MMP-9 mRNA levels, an enzyme required for the degradation of ECM, and thereby metastasis. We suggest that the reduced level of MMP-9, after LQB-118 treatment in PC3 cells, must result from the augmented levels of Reck, a matrix metalloprotease inhibitor.

The observed effects of LQB-118 in this paper are extremely important since PCa cells have a highly invasive behavior. These results permitted us to improve the understanding of the LQB-118 antitumor effects, since the inhibition of Akt phosphorylation (Ser473) seems to be involved not only on inhibition of cell migration/invasion, as demonstrated here, but also on the inhibition of tumor cell proliferation and apoptosis induction (33), effects already demonstrated for LQB-118 in prostate cancer (10) and leukemia cells (34). In addition, it is important to note that experiments demonstrating the LQB-118 in vivo antitumor efficacy in prostate cancer (11), Erlich solid tumors and B16F10 melanoma cells (35) have already been performed, and safety preclinical studies with LQB-118 have not shown immunotoxicity (35) or subacute toxicity (36).

Conclusion

Collectively, our findings provide new insights into the molecular mechanism underlying LQB-118-induced anti-migration/invasion effects on metastatic PCa cells, partly by inhibiting the Akt/GSK3β phosphorylation, a key pathway for migration, invasion, and proliferation of tumor cells. Herein, we demonstrated that LQB-118 mediates these effects by an αvβ3/FAK-independent pathway, highlighting a potential role in blocking the epithelial-mesenchymal transition of cancer cells towards a metastatic phenotype. Furthermore, LQB-118 seems to modulate the invasiveness behavior reducing the metalloprotease MMP-9 expression, which can result from up-regulation of its inhibitor Reck. LQB-118 could therefore be part of a promising new strategy for anticancer therapy.

Acknowledgements

The Authors thank the LIA-BPPN Laboratory technicians for their excellent assistance. This work was supported by Grants from FAPERJ and CNPq, Brazil.

Footnotes

  • Authors’ Contributions

    Research design: Martino T, Sabino KCC; Experimental work: Martino, TM; LQB-118 synthesis and supply: Costa PRR and Chaquip Neto; Western Blot expertise and execution: Martino T, de Bem GF, Resende AC; Cytofluorimetric expertise and execution: Martino T, Justo G; Original manuscript writing: Martino T and Sabino KCC; Result analysis and discussion: Martino T, Sabino KCC, Justo G, Coelho, MGP, Netto C, Costa PRR. Project administration: Sabino KCC; Justo G, Coelho MGP; Review and editing: Martino T, Coelho MGP and Sabino KCC. Checking and submission: Justo G. All Authors read and approved the final draft.

  • Conflicts of Interest

    The Authors have no conflicts of interest to declare in relation to this study.

  • Received October 29, 2022.
  • Revision received November 9, 2022.
  • Accepted November 11, 2022.

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LQB-118 Suppresses Migration and Invasion of Prostate Cancer Cells by Modulating the Akt/GSK3β Pathway and MMP-9/Reck Gene Expression
THIAGO MARTINO, GRAZIELE FREITAS DE BEM, SHIRLEY VANIA MOURA SANTOS, MARSEN GARCIA PINTO COELHO, ANGELA DE CASTRO RESENDE, CHAQUIP NETTO, PAULO ROBERTO RIBEIRO COSTA, GRAÇA JUSTO, KATIA COSTA DE CARVALHO SABINO
Anticancer Research Jan 2023, 43 (1) 359-367; DOI: 10.21873/anticanres.16171
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