Semi-synthetic sapogenin derivatives inhibit inflammation-induced tumorigenic signaling alterations in prostate carcinogenesis

doi:10.21203/rs.3.rs-4841687/v1

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Research Article

Semi-synthetic sapogenin derivatives inhibit inflammation-induced tumorigenic signaling alterations in prostate carcinogenesis

https://doi.org/10.21203/rs.3.rs-4841687/v1

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Purpose

Prostatic inflammation is closely linked to prostate cancer (PCa) and plays a pivotal role in tumor development and progression via altering a wide range of cellular mechanisms, including proliferation, metastasis, and angiogenesis. Since the tumor infiltration of the immune system cells, such as macrophages, leads to higher expression of inflammatory mediators in the tumor microenvironment, the use of anti-inflammatory drugs could provide a valid contribution to preventing and treating cancer. In our research, we explored semi-synthetic derivatives of cycloastragenol (CA) and astragenol (AG) molecules to assess their potential to inhibit inflammation-induced tumorigenic signaling pathways.

Methods

Levels of reactive oxygen species (ROS) and nitric oxide (NO), expression of proteins function in tumorigenic cellular mechanisms, cellular apoptosis and migration were examined.

Results

Building on our previous study, which revealed the molecules' inhibitory activity on the nuclear factor kappa B (NF-κB), we discovered that these molecules also suppressed enhanced cell proliferation and migration by inhibiting distinct molecular mechanisms. Additionally, they were able to mitigate elevated levels of ROS, NO, and the expression of vascular endothelial growth factor (VEGF) caused by inflammation. Moreover, the anti-inflammatory properties of these molecules partially restored the loss of androgen receptor (AR) and tumor suppressor NKX3.1, both of which contribute to prostate tumorigenesis in an inflammatory microenvironment. Furthermore, they reversed the activation of Akt and β-catenin signaling induced by inflammation, suggesting their potential to inhibit inflammation-related prostate tumorigenesis. Our study also demonstrated that the molecules exhibited dose-dependent effects on inducing cell cycle arrest and apoptosis, as evidenced by increased p21 and decreased BCL-2 protein levels, ultimately leading to activated cell death and suppressed cellular migration.

Conclusion

Semi-synthetic sapogenol derivatives have displayed promise as anti-inflammatory and anticancer drug candidates for targeting prostatic inflammation and inflammation-induced prostate carcinogenesis.

Cycloastragenol

astragenol

prostate cancer

β-catenin

inflammation-induced tumorigenesis

CA and AG derivatives inhibit inflammation-induced prostate carcinogenesis.
These derivatives exhibit concentration-dependent anti-inflammatory and cytotoxic activity.
They reduce inflammation-induced ROS and NO release.
They repress tumorigenic β-catenin activation in inflammatory tumor microenvironment.
They promote apoptosis and suppress cellular migration.

Inflammation is reported as one of the major contributors to prostate carcinogenesis (Tewari et al., 2018, Catalano et al., 2023). Recent literature has shown correlations between soluble inflammatory mediators, such as cytokines, revealing a direct relationship between inflammation and prostate cancer. During chronic inflammation, cytokines promote tumorigenesis via activated proliferative and migrative signaling pathways in addition to the damaging effects of reactive oxygen and nitrogen species (RONS) generated during inflammation (Haverkamp et al., 2008, Archer et al., 2020).

In vitro and in vivo cancer models have demonstrated that chronic inflammation alters signal transduction pathways (Ho et al., 2004), including Protein kinase B(Akt) and Wnt/β-catenin inducing neoplastic transformation in the prostate tissue (Chesire and Isaacs, 2003). Macrophages activated by inflammation secrete many Wnt glycoproteins, leading to transcriptional activation of β-catenin. Sustained activation of the Wnt signaling pathway and accumulation of β-catenin is frequently observed in solid tumors and results in uncontrolled cell growth and differentiation (Chesire and Isaacs, 2003, Tewari et al., 2021). Inflammatory tumor microenvironment also induces proteasomal degradation of AR (Debelec-Butuner et al., 2012). Prostate cells require AR for proliferation, and therefore, androgen ablation therapy initially causes tumors to shrink. However, it also accelerates the transition to the AR-independent phase of PCa, triggering androgen-independent activation of many cellular pathways that control survival and growth, including PI3K/Akt. These pathways eliminate the AR dependency of the cells for proliferation, and increased activity of the phosphoinositide 3-kinase (PI3K)/Akt pathway further enhances tumor progression (Murillo et al., 2001).

NKX3.1 is an AR-regulated tumor suppressor, and its ubiquitination induced by inflammatory cytokines IL-1β and TNF-α occurs during prostatic inflammatory atrophy (Markowski et al., 2008). Loss of NKX3.1 was shown to lead to neoplastic transformation of prostate cells and NKX3.1 expression has been found to decrease at sites of inflammatory atrophy and preinvasive prostate cancer (Bowen et al., 2000, Khalili et al., 2010).

RONS-induced tumorigenic mechanisms also need to be considered as therapeutic targets of inflammation-related carcinogenesis. ROS formation rather than ROS accumulation triggers tumorigenic mechanisms. Therefore, simply neutralizing ROS within the cell, the way antioxidants act, is not sufficient to protect the cell, and further treatment strategies indirectly reducing ROS formation, such as anti-inflammatory treatment, are essential for the inhibition of ROS-related tumorigenic mechanisms (Khandrika et al., 2009). Moreover, exposure to nitric oxide leads to post-translational modifications of the tumor suppressor gene p53, resulting in its functional loss. p53 plays a role in the inflammatory response by regulating the expression of certain genes, including NOS2. In chronic inflammation, NO• both activate the p53 tumor suppressor pathway and induce oncogenic mutations in the p53 gene (Hussain and Harris, 2007). Consequently, the effect of the drug candidates on NO• release also needs to be considered as a therapeutic target in the inflammatory microenvironment (Catalano et al., 2023).

Anti-inflammatory therapeutics have been known as a chemopreventive tool for inflammation-related carcinogenesis. Long-term use of NSAIDs is determined to correlate to decreased cancer risk and incidence for prostate, colon, stomach, breast, and lung cancer cases (Kismet et al., 2004, Chan and Detering, 2013, Kopp et al., 2013, Tinsley et al., 2013). Experimental and clinical studies revealed that anti-inflammatory agents generally show their anticancer effect via inhibiting cellular pathways such as NFκB. Besides, suppression of ROS levels due to the regular use of antioxidants was also determined to be effective in reducing prostate cancer incidence and mortality (Schetter et al., 2010).

Anti-inflammatory and anticancer activities of natural products are demonstrated in drug development studies. Semi-synthetic derivatives of secondary metabolites can provide structural diversity and better activity. Saponins are polar glycosylates comprised of triterpene or steroid aglycone with one or more sugar chains. They show a wide array of biological activities, including antimicrobial, antifungal, antioxidant, anti-inflammatory, immunomodulatory, antiviral, antitumoral, and neuroprotective properties, as documented in the literature (Sparg et al., 2004, Osbourn et al., 2011, Uner et al., 2022, Cotrim Ribeiro et al., 2024, Li et al., 2024). Triterpenoids play a role in the regulation of inflammation through their anti-inflammatory properties. Triterpene saponins from Astragalus sp. are also determined to have anti-inflammatory and anticancer activity (Dong et al., 2019, Uner et al., 2020, Ionkova et al., 2022, Dong et al., 2023) promoting further studies to discover new natural and semi-synthetic molecules as drug candidates.

The following compounds - C2, C30, C31, A17, and A29 - are semi-synthetic derivatives of cycloastragenol (CA) and astragenol (AG). They have been identified as potential anti-inflammatory agents due to their ability to inhibit NFκB, COX2, and PGE2 (Debelec-Butuner et al., 2018). This study aimed to further investigate their inhibitory effects on inflammation-related signaling mechanisms, which play a role in promoting cell proliferation, migration, and angiogenesis in prostate carcinogenesis.

2.1. Derivatization of cycloastragenol and astragenol

Cycloastragenol (CA): CA (98% purity) was donated by Bionorm Natural Products (Turkey).

Astragenol (AG): AG was synthesized from 2.0 mmol of CA, which was dissolved in MeOH (50 mL) and then treated with conc. H₂SO₄ (5 mL). The whole mixture was heated under reflux for 6h. The reaction mixture was poured into water and extracted with EtOAc. The EtOAc extracts were washed successively with sat. NaHCO₃ aq. then water, and dried over Na₂SO₄ and filtered. The filtrates were evaporated, and chromatographic separation of the crude products on silica gel columns was performed by using n-hexane:EtOAc (6:4) to give AG.

A17: To a solution of AG (50 mg, 0.102 mmol) in pyridine (5 mL) at 25°Cwas added acetic anhydride (250 µL). The reaction mixture was stirred at room temperature for 12h, quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The organic layers were dried over Na₂SO₄ and concentrated. The residue was then purified through column chromatography (n-hexane: EtOAc; 8:2) to produce A17.

A29: To a stirring solution of AG (100 mg, 0.02 mmol) in acetone (100 mL), the Jones reagent (0.4 mL, 3.3 mmol) was added slowly on the ice bath and stirred till completion of the reaction. After adding sat. NaHCO₃ aq., the aqueous mixture was extracted with CH₂Cl₂ (3 × 25mL). The combined organic layers were washed with brine solution, dried over Na₂SO₄, and filtered. The filtrate was evaporated, and chromatographic separation of the crude product on a silica gel column was performed by using hexanes:ethyl acetate (8:2) as eluent to give A5. A5 (120 mg) was dissolved in 8 mL THF and was added NaOMe (135 mg) at room temperature. The reaction mixture was stirred overnight and added sat. aq. NaHCO₃, then the aqueous mixture was extracted with CH₂Cl₂ (3 × 25mL). The combined organic layers were washed with brine solution, dried over Na₂SO₄, and filtered. The filtrate was evaporated, and chromatographic separation of the crude product on a silica gel column was performed by using n-hexane:EtOAc (9:1) as an eluent to give A29.

C2: 400 µL propionic anhydride (3.12 mmol) was added dropwise to a solution of CA (100 mg, 0.203 mmol) and 5 mg DMAP (0.04 mmol) in pyridine (10.0 mL). The reaction mixture was heated under reflux for 12 h. The solution of completed reaction was added to the cold 1 M HCl solution and was extracted four times with 25 mL chloroform. Combined organic layers were dried over Na₂SO₄ and filtered. The filtered organic layer was evaporated under vacuum, subjected to a silica gel flash chromatography, and purified with hexanes-EtOAc (4:1) to afford C2.

C30: 200 µL acetic anhydride (2.112 mmol) was added dropwise to a solution of CA (100 mg, 0.203 mmol) and 5 mg DMAP (0.040 mmol) in pyridine (10.0 mL). The mixture was heated under reflux for 12 h. The reaction mixture was added to the cold 1M HCl solution and extracted four times with 25 mL chloroform. Combined organic layers were dried over Na₂SO₄ and filtered. The filtered organic layer was evaporated under vacuum, subjected to a silica gel flash chromatography, and purified with hexanes-EtOAc (7:3) to give C30.

C31: 100 µL acetic anhydride (1.056 mmol) was added to the solution of CA (100 mg, 0.203 mmol) and 3 mg DMAP (0.024 mmol) in pyridine (10.0 mL). The reaction mixture was heated under reflux for 4 h. The reaction solution was quenched by adding cold 1 M HCl solution and extracted four times with 25 mL chloroform. Combined organic layers were dried over Na₂SO₄, filtered, and evaporated under a vacuum. The reaction extract was subjected to a silica gel flash chromatography and purified with hexanes-EtOAc (5:5) to give the pure compound C31.

2.2. Cell culture and treatments

LNCaP, U937, RAW264.7, and PC-3 cells were obtained from the American Type Culture Collection (Manassas, VA). LNCaP, U937 and RAW264.7 (RPMI 1640) and PC3 (DMEM-F12) (Gibco-Invitrogen, US) were propagated using indicated mediums supplemented with 10% FBS, L-glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 mg/ mL) at 37°C with 5% CO₂. Conditioned media (CM/containing 125 pg/mL of TNF-α) treatments were performed. Treatments with N-acetyl-L-cysteine (L-NAC; 10 mM), lipopolysaccharide (LPS; 1 µg/ml), parthenolide (4 µM), and Bay11-7082 (5 µM) were performed at indicated concentrations. Molecule treatments were performed 2 h before either CM or LPS treatments.

2.3. Preparation of conditioned media (CM) and detection of TNF-α concentration in CM

Macrophage differentiation and cytokine production were performed by using U937 cells according to the previous protocols. TNF-α levels were analyzed using an ELISA according to the manufacturer’s instructions (Invitrogen, US). TNF-α was selected as a measure of CM concentration based on previous studies (Debelec-Butuner et al., 2012, Debelec-Butuner et al., 2014). The concentration of TNF-α in CM treatments was adjusted by diluting the CM with a normal medium before application to LNCaP cells.

2.4. Intracellular ROS measurement by H2DCFDA assay

LNCaP cells (10⁴/well) were seeded into 96-well plates and incubated for 48 hours. The cells were incubated with 10 µM DCFH-DA (2′ 7′-dichlorodihydrofluorescein diacetate, Molecular Probes, US) in total medium without phenol red for 30 min at 37°C. After DCFH-DA was removed, the cells were gently washed using a phenol-red-free medium. Then, the treatments were performed and the fluorescence intensity was measured every 20 min at 37°C for up to 4 h using a Fluoroskan microplate reader (Thermo Fisher Scientific, US).

2.5. Measurement of NO release

5x10⁴/well RAW264.7 cells were seeded in a 96-well plate and incubated for 24 hours. Then, the medium was changed to phenol red-free medium and treatments were performed for 24 hours. The supernatant was collected and centrifuged at 300g for 5 min. 35 µl of supernatant was transferred to a new 96-well plate, and the same volume of modified Griess reagent (Sigma-Aldrich) at room temperature was added and mixed by pipetting. It was incubated in the dark for 15 min. Absorbance was measured at 540/630 nm and the results are calculated as the relative level of NO release compared to control cells.

2.6. Protein extraction and western blotting

Cells were grown in 6-cm plates (Sarstedt, Germany), and after treatments ended, cells were washed once with PBS before cell lysis. Cells with 250 µl ice-cold modified RIPA buffer (10 mM Tris Cl (pH 8.0), 1% Triton X-100, 0.1% SDS, 0.1% Na deoxycholate, 1 mM EDTA, 1 mM EGTA and 140 mM NaCl) containing both protease and phosphatase inhibitors were sonicated for 20 seconds (25% power, 0.5 cycles), centrifuged at 12000 g for 10 min at 4°C, and the cleared supernatants were obtained. The protein concentration was determined using a BCA assay (Sigma, UK). Western blots were performed under standard conditions using 50 µg of protein lysate separated on a 10–12% SDS-PAGE gel and transferred to a PVDF membrane (Amersham, UK) using a wet transfer blotter. The PVDF membrane was blocked with 5% dry milk in TBS-T (Tris-Borate-Saline solution containing 0.1% Tween 20), and then primary and secondary antibody incubations were performed using TBS-T containing 0.5% dry milk or 5% BSA at RT for 1 h or at 4°C overnight. The membranes were developed using ECL plus reagent (Amersham, UK) for 5 min, and bands were detected for the following antibodies, which were purchased and used according to the manufacturer’s recommendations: AR (Millipore); NKX3.1; p53; VEGF; p-AKT(S473), Akt1, p-β-catenin(S552), β-catenin, E-cadherin, p21, pan-acetyl; ac-H4(K5,8,12,16) (SCBT), BCL2 (Cell Signaling), GAPDH (Ambion); β-actin (Sigma).

2.7. Detection of apoptosis by flow-cytometry Annexin V/PI staining

2.5x10⁵ cells were grown in 6-well plates (Sarstedt, Germany), and after treatments ended, cells were collected by trypsinization. Cells were processed as recommended in BD Pharmingen FITC Annexin V apoptosis detection kit I. Briefly, cells were washed with cold PBS twice and resuspended in 100 ul 1x binding buffer. 2 ul Annexin V and 2 ul PI were added to cells and incubated for 15 min in the dark at room temperature. 400 ul 1x binding buffer was added to cells, and samples were analyzed by flow cytometry (BD Accuri C6).

2.8. Scratch assay

2.5x10⁵ cells were grown in 6-well plates (Sarstedt, Germany). Cells were scraped by using a micropipette tip and washed with PBS. Treatments were performed in fresh medium, and phase-contrast photos were captured at 0, 24, and 48 h. Vertical lines defining cell boundaries were added to the photographs and the distance between these lines was analyzed by using Image J software. Relative migrations of control and treated cells were calculated by normalization to 0. h for each sample and presented as %migration.

2.9. Statistics

Data are presented as means ± standard deviation (SD). Student’s t-test with two-tailed equal variance was used to assess the statistical significance between pairs as *p < 0.05, *p < 0.01, *p < 0.001.

Structures of CA, AG, and the semi-synthesis of their derivatives C2, C3, C31, A17, and A29 are shown in Fig. 1.

3.1. Suppression of intracellular reactive oxygen species

Prostatic inflammation leads to an increase in intracellular ROS levels through the activation of transcriptionally induced pro-oxidant genes. As the anti-inflammatory effect of the molecules presumably contributes to the inhibition of ROS, intracellular ROS levels in LNCaP cells upon molecule treatments were measured kinetically for up to 4 h by the H2DCFDA method. It was determined that the increased ROS level due to the treatment of 100 µM H₂O₂ was suppressed by the CA- and AG-derived compounds. LNAC served as a positive control for the measurement of ROS inhibition and resulted in a suppression below the basal ROS level. Further, the inhibitory effect of the test compounds was comparable to BAY-117082, an NFκB inhibitor, suggesting that the effect should be mediated by NFkB inhibition instead of direct ROS neutralization (Fig. 2A and B).

3.2. Inhibition of NO release

The inhibitory effects of the molecules on iNOS activation were investigated by measurement of the level of NO release in RAW264.7 macrophage cells using the Griess method. NO release was found to increase 44.6-fold due to LPS treatment (1 µg/ml, 24 hours) and inhibited 6.2-fold with parthenolide (4 µM) used as a positive control. It was determined that astragenol derivatives A17 and A29 exhibited 5.1- and 36.1-fold while cycloastragenol derivatives C2, C30, and C31 showed 22.1, 22.2, and 13.7 relative fold change of NO levels sequentially. BAY-11708 reduced the NO level to 10.4-fold, and parthenolide, another NFκB inhibitor, suppressed it to 6.2-fold (Fig. 3). Since NO release has an immunoregulatory effect by inducing cytokine and chemokine release from macrophages and neutrophils, lowering the NO level will also inhibit macrophage infiltration and cytokine release in epithelial cells, which is eventually related to inflammation-induced carcinogenic alterations (Kobayashi, 2010). Therefore, it was concluded that A17 and C31, in particular, have a high potential for preventing nitrosative protein and DNA damage (Murata et al., 2012).

3.3. Effects of the molecules on prostate-specific carcinogenesis pathways

Studies on the inflammation-related prostatic carcinogenesis mechanisms have proven that proteasomal degradation of the proteins androgen receptor (AR) and the tumor suppressor NKX3.1, which function on prostate cell survival, are induced in the prostatic inflammatory microenvironment (Khalili et al., 2010, Debelec-Butuner et al., 2012). In addition, functional loss of p53 is known to lead to cancer initiation. Therefore, to investigate the effect of the molecules on AR, NKX3.1, and p53 protein levels under inflammatory conditions, LNCaP cells were grown in a conditioned medium containing 125 pg/ml TNF-α with further molecule treatments performed at their IC₂₀ concentrations for 24 h. Reduced protein levels of AR, NKX3.1, and p53 were observed as a result of CM treatment, consistent with our previous studies (Debelec-Butuner et al., 2012). It was determined that A29 provided a slight recovery in AR and NKX3.1 protein levels, while C30 was efficient in inhibiting the loss of p53 (Fig. 4).

As no significant recovery in AR and NKX3.1 protein levels likely resulted from the cytotoxic effects of the molecules on survival signaling pathways in addition to NFκB inhibition (Debelec-Butuner et al., 2018), treatments were performed by using IC₅ to alleviate the cytotoxic effect. Inflammation-induced proteasomal degradations of AR and NKX3.1 were found to be reversed by A17, A29, and C2. C31 provided a partial recovery at the NKX3.1 protein level. (Fig. 4).

3.4. Inhibition of the inflammation-induced VEGF expression

Enhanced expression of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, is a critical stage of tumorigenesis. VEGF expression levels were investigated in inflammatory conditions following a 2-h pre-treatment of the molecules. LNCaP cells were treated with 5 µg/ml LPS along with the test compounds for 48 h. No change was detected in the VEGF protein level due to the effect of either LPS or the compounds (Fig. 5). To mimic the enhanced VEGF expression in the inflammatory tumor microenvironment, cells were treated with CM containing 125 pg/ml TNF-α for 24 h w/w.o. the molecules. It was determined that increased VEGF levels due to inflammatory media treatment were completely suppressed by the candidate molecules (Fig. 5).

3.5. Inhibitory effects on tumorigenic β-catenin signaling

The effect of the molecules on the β-catenin signaling pathway, which significantly contributes to inflammation-induced carcinogenesis, was investigated by treatment of LNCaP cells with 48 h of 5 µg/ml LPS following a 2-h pre-treatment of the compounds. It was determined that Akt(S473) phosphorylation, playing a major role in enhanced cell proliferation, was inhibited by all compounds, revealing that A29 and C31 were the most active ones. In addition, β-catenin expression increased due to LPS was suppressed by A29, C2, C30, and C31. It was observed that there was a slight decrease in E-cadherin level due to LPS application, and A29 especially reversed this effect. GAPDH labeling was used as a loading control (Fig. 6A and B).

Increased Akt(S473) phosphorylation due to inflammatory CM treatment was detected, consistent with our previous studies (Debelec-Butuner et al., 2014). Immunoblotting results revealed that the molecules reversed this proliferative activation to basal levels. Further, the suppression of Akt activity, which is responsible for the phosphorylation of β-catenin at S552, resulted in a decrease in CM-induced p-β-catenin(S552). Similar to the results shown in the presence of LPS, enhanced β-catenin expression level by CM was observed to be suppressed exhibiting A17, A29, and C31 are the most efficient candidates on the tumorigenic β-catenin pathway (Fig. 6C and D). As inhibition of S552 phosphorylation of β-catenin reduces its nuclear translocation, these drug candidates were expected to consequently inhibit the expression of β-catenin target genes, which mediate its proliferative and migrative effects. Therefore, it was concluded that inhibition of the p-β-catenin(S552) level increased the antiproliferative and antimigrative potential of the molecules, leading us to analyze the ultimate cellular effect of the molecules on cell death and migration.

3.6. Induction of apoptotic cell death as an anticancer drug candidate

Our results demonstrating the inhibitory effects of the molecules on the proliferative pAkt/β-catenin signaling suggested the cell death mechanisms also might be stimulated in a concentration-dependent manner. To investigate the effects of the compounds on apoptotic signaling, LNCaP cells were treated with the molecules at IC₂₀ and IC₅₀ concentrations for 24 h. Protein acetylation levels were also examined, considering whether the molecules could show their activity through the acyl group in their structure via transacylation reactions. As a result of pan-acetyl labeling, which shows the global acetylation level in proteins, it was detected that all molecules enhanced global acetylation levels. As a result of specific labeling for lysine (K) 5, 8, 12, and 16, acetylations of histone4 (H4), A17, A29, and C2 were found to enhance H4 acetylation.

Moreover, p21 expression, which is responsible for cell cycle control, stops the cell cycle so that the damaged cell does not continue to divide and activates either DNA repair mechanisms or cell death. p21 level was observed to be increased with a negative correlation to molecule concentrations, suggesting that IC₂₀ concentrations of the molecules might lead cells to activate the damage repair, while higher concentrations of the molecules might alleviate the p21 increase and induce cell death rather than damage repair. Consistent with concentration-dependent p21 alteration, the antiapoptotic BCL2 protein level did not change upon IC₂₀ of A17, A29, and C30 but was suppressed at IC₅₀ concentrations. C2 treatments were observed not to affect the BCL2 level. However, C31 treatments resulted in a decrease in comparison to the control level (Fig. 7A and B).

To measure the potential of the molecules on apoptotic cell death activation, LNCaP and PC3 cells were treated with IC₅₀ concentrations of the molecules for 24 h. Early and late apoptotic cell rates were determined by Annexin V/PI labeling in flow cytometry and presented as %apoptosis in comparison to control. Treatment of PC3 cells demonstrated that apoptosis was triggered in approximately half of the population by C30 and C31, in 30% of the cells by C2, and in 15% by A17 and A29. In addition, although it seems that there was no significant increase in the rate of apoptotic population in LNCaP cells with a slight induction due to C30 and C31, at 6% and 10%, respectively, the increased rate of death cells in the UL region of the flow-cytometry dot plot graphs implies that the molecules severely induced cell death (Fig. 7 and Suppl Fig. 1).

3.7. Inhibitory effects of the molecules in cellular migration

Current results revealing the inhibitory effect on β-catenin signaling and previous results on NFkB inhibition (Debelec-Butuner et al., 2018) led us to investigate cellular migration, and scratch assay was performed upon molecule treatments at their anti-inflammatory concentrations in LNCaP and PC3 cells.

Scratched areas were pre-marked and photographed at 0, 24, and 48 h. Image analysis was performed by using Image J and the change in cellular migration was plotted as %migration relative to control cells. Results revealed that the compounds significantly suppressed cellular migration in a time-dependent manner. It is determined that the migration rate decreased to approximately 50% at the 24th h and 30% at the 48th h in LNCaPs. PC3 cells were detected to be more resistant, exhibiting 60% and 40% migration rates (Fig. 8 and Suppl Fig. 2). Overall results revealed that cellular migration was significantly suppressed by the tested molecules in a time-dependent manner.

Epidemiological data indicates that prostate cancer incidence is significantly high in developed countries and extensively depends on environmental factors. Prostatic inflammation is a leading cause of prostate cancer, and environmental factors enhance inflammatory diseases of prostate tissue (Oseni et al., 2023). Therefore, inhibition of inflammation-related tumorigenic signaling pathways, which function in cell proliferation and migration, should be considered essential for both cancer chemoprevention and anticancer drug development strategies (Nambiar and Singh, 2013). In this study, we investigated the inhibitory activity of the phytochemical derived analogs mentioned above on inflammation-induced tumorigenic alterations. As their anti-inflammatory effects have been demonstrated in our previous study, they are suggested as potential anticancer drug candidates in the suppression of molecular targets that mediate proliferation and migration in inflammatory tumor microenvironments. Inhibition of NFkB signaling was determined to result in suppressed transcriptional activation of p65, decreased PGE2 release, and cell proliferation ultimately.

ROS and NO produced during chronic inflammation lead to DNA and protein damage, which are leading factors of oncogenic mutations and tumor heterogeneity. Therefore, changes in their levels were also investigated and found to be alleviated by the molecules. In addition, our compounds applied at their anti-inflammatory concentrations showed a similar suppression rate to the NFkB inhibitor, differently from antioxidant LNAC. As the suppression rate upon molecule treatments did not decrease the ROS level lower than the control, we concluded that the molecules inhibit H₂O₂-induced ROS formation instead of neutralizing. Since the therapeutic molecules that inhibit ROS formation are more efficient than the ROS scavenging/neutralizing molecules in oxidative stress-related carcinogenesis (Khandrika et al., 2009, Reuter et al., 2010, Morgan and Liu, 2011), tested molecules inhibiting ROS formation, possibly through NFkB inhibition, are concluded to be potential drug candidates for cancer prevention. In addition, as NO release by pro-inflammatory cells such as tumor-associated macrophages is a major cause of further tumorigenic cellular damage, the efficiency of molecules in decreasing LPS-induced NO levels enhances their anticancer potential.

Moreover, inflammation-induced protein losses of AR and NKX3.1 were detected to be partially recovered. Although AR function stimulates prostate cell proliferation, androgen-ablation therapy was elicited to facilitate the castration-resistant stage of PCa, possibly because of its regulatory role in oxidative stress regulation and antioxidant response. Additionally, increased VEGF expression by inflammatory conditioned media was significantly suppressed, showing the anti-angiogenic effect of the molecules. Therefore, protection of the cell from AR and NKX3.1 loss and increased VEGF expression at relatively lower concentrations of the molecules support their anti-inflammatory potential in cancer chemoprevention.

Inflammation-induced activation of Akt and β-catenin was simulated by LPS and inflammatory conditioned media treatments, and enhanced activations were determined to be repressed. As these factors have intense crosstalk with other signaling pathways such as NFkB, target of rapamycin in mammals (mTOR), mitogen-activated protein kinase (MAPK), extracellular signaling regulated kinase (ERK), and glycogen synthase kinase-3 beta (GSK-3β) (Vallee and Lecarpentier, 2018, Cui et al., 2020, Daisy Precilla et al., 2022), which function in proliferative, migration, and cell death, inhibitory effects of molecules are encouraging for investigating ultimate cellular alterations in cancer cells.

Increased p21 and reduced antiapoptotic BCL2 levels proved that the molecules also contribute to cell cycle transition and induce apoptosis in androgen-responsive prostate cancer cell line LNCaP and castration-resistant PC-3 cells. Inhibited cell migration, possibly due to repression of β-catenin signaling at IC₅₀ concentration of the molecules, confirmed their anticancer potential. However, as the increase in histone acetylation after molecule treatments could be mediated by histone deacetylase (HDAC) inhibition or histone acetylase (HAT) activation, further experiments are required to reveal whether the molecules induce protein acylation by interacting with HDAC/HAT or by directly transferring acyl groups to proteins (Villagra et al., 2010).

Overall, evaluation of survival and anti-inflammatory effects at IC₅ and IC₂₀ concentrations and apoptotic/anti-migrative effects at IC₅₀ concentrations led us to claim that concentration-dependent cellular effects of the molecules on inflammation-induced tumorigenic mechanisms showed their potential as a therapeutic candidate both for cancer chemoprevention and anticancer therapy.

Chronic inflammation has a leading role in all three stages of prostate carcinogenesis, namely initiation, promotion, and progression. NSAIDs have been shown to have both a preventive and therapeutic potential in prostate cancer by regulating the signaling molecules, including the ones investigated in this study, such as COX-2, p53, p21, and Bcl-2, and thus contributing to the cellular mechanisms of the cell cycle arrest, apoptosis, cell growth, metastasis, and angiogenesis (Maghsoudi et al., 2023).

The utilization of natural products in cancer treatment has become attractive as specific secondary metabolites can be efficient on specific therapeutic targets within signaling pathways and molecular processes involved in cancer development. Secondly, the administration of these compounds at appropriate doses and forms mostly exhibits fewer adverse effects in comparison to conventional cancer treatments. Therefore, they can be involved in combination therapies with chemotherapy to improve the effectiveness of the therapy and to alleviate the adverse effects.

Natural compounds in a wide range of structures have been known to be beneficial in prostate cancer treatment (Fernandes et al., 2023). Geraniol, an anti-inflammatory natural monoterpene, has been known to modulate inflammation and induce cell cycle arrest and apoptosis and, therefore, is a potential anticancer candidate for inflammation-related cancers, including colon, prostate, breast, lung, skin, kidney, liver, and pancreatic cancer (Ben Ammar, 2023). β-sitosterol, a phytosterol structurally similar to cholesterol, has been shown to interfere with multiple signaling pathways involved in inflammation, cell growth, apoptosis, metastasis, and angiogenesis, exhibiting anticancer activities on various cancer types accordingly (Wang et al., 2023, Nandi et al., 2024). Similarly, Wang et al. demonstrated that Astragalus saponins suppressed LPS-induced NO generation in mouse macrophages and inhibited cellular growth in promyelocytic leukemic HL-60 cells and T-lymphocyte leukemic Jurkat cells. In addition, their combination with the chemotherapeutic drug 5-FU was observed to reverse the hematologic toxicities that 5-FU causes, such as reduced white blood cells and decreased hematocrit (Wang et al., 2016).

Astragaloside IV, a saponoside of cycloartane-type, was intensely investigated, revealing its various pharmacological activities, including anti-inflammatory, antioxidant, anticancer, immunomodulatory, and neuroprotective (Zhang et al., 2020). Further, it was particularly shown to inhibit cell growth and induce apoptosis in prostate cancer cells (Tan et al., 2018) and LPS-activated Wnt/β-catenin signaling with related inflammatory cytokines and oxidative factors in bovine mammary epithelial cells (Fan et al., 2022). In the study of Yan et al., triterpenoid saponins were shown to exert anti-inflammatory and anti-apoptotic effects through autophagy by reducing TNF-α-induced inflammation and apoptosis and activating autophagy in HUVECs (Yan et al., 2022).

We previously demonstrated the anti-inflammatory bioactivity of our semi-synthetic cycloartane-type triterpene analogs in the prostate cancer concept. Inflammation is strongly associated with prostate cancer and plays a key role in tumor development and progression. Therefore, repression of inflammation-related molecular alterations is a useful strategy for cancer therapy. In this study, our results revealed that these molecules inhibit inflammation-induced tumorigenic signaling at their anti-inflammatory concentrations and trigger cell death at higher concentrations, supporting their efficiency in inhibiting inflammation-related carcinogenesis and cancer therapy. In addition, targeting inflammatory signaling pathways, either alone or in combination with chemotherapeutic agents, can be beneficial for the prevention and treatment of prostate cancer, making these compounds potent candidates for combinational therapy approaches (Maghsoudi et al., 2023).

Anti-inflammatory drug candidates can be beneficial in the suppression of inflammation-induced carcinogenesis and should be evaluated as drug candidates not only for cancer chemoprevention but also for anticancer therapy in cancer promotion and progression stages in the inflammatory microenvironment of cancerous tissue. Semi-synthetic sapogenin derivatives, determined to be efficient in inhibiting inflammation-induced tumorigenic alterations, are potent anti-inflammatory and anticancer drug candidates for prostate cancer treatment, encouraging further studies on their potential in inflammation-related cancer types.

Human Ethics and Consent to Participate

Not applicable.

Competing Interests:

The authors declare no competing financial interest.

Funding:

This work was supported by the Scientific and Technological Research Council of Türkiye (TUBITAK) with project number 113Z078 to BDB.

Author Contribution

B.DB designed the study, O.T. IH.A. and E.B. provided molecule derivatives, B.DB and MB.O. performed molecular experiments, collected the data, and analyzed the data; B.DB. and E.B. wrote the manuscript; all authors critically reviewed the manuscript.

Data Availability

Data will be made available on request.

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Semi-synthetic sapogenin derivatives inhibit inflammation-induced tumorigenic signaling alterations in prostate carcinogenesis

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Abstract

Figures

Highlights

1. Introduction

2. Materials and methods

2.1. Derivatization of cycloastragenol and astragenol

2.2. Cell culture and treatments

2.3. Preparation of conditioned media (CM) and detection of TNF-α concentration in CM

2.4. Intracellular ROS measurement by H2DCFDA assay

2.5. Measurement of NO release

2.6. Protein extraction and western blotting

2.7. Detection of apoptosis by flow-cytometry Annexin V/PI staining

2.8. Scratch assay

2.9. Statistics

3. Results

3.1. Suppression of intracellular reactive oxygen species

3.2. Inhibition of NO release

3.3. Effects of the molecules on prostate-specific carcinogenesis pathways

3.4. Inhibition of the inflammation-induced VEGF expression

3.5. Inhibitory effects on tumorigenic β-catenin signaling

3.6. Induction of apoptotic cell death as an anticancer drug candidate

3.7. Inhibitory effects of the molecules in cellular migration

4. Discussion

5. Conclusion

Declarations

Competing Interests:

Funding:

Author Contribution

Data Availability

References

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Supplementary Files

Status:

Version 1