Antitumor activity and mechanism of action of S-adenosylmethionine in prostate cancer cells

Prostate cancer (PCa) is the most common male malignancy in Western countries, with approximately 1.6 million new cases annually. The etiology of PCa is not yet fully understood, but the main causes are attributable to environmental and genetic factors. Because of the fundamental role in tumor progression of the androgen receptor, hormonal therapy or androgen deprivation therapy induces tumor regression and apoptosis in hormone-sensitive cells. However, metastatic castration-resistant prostate cancer (mCRPC), an advanced stage of prostate cancer, does not respond to conventional therapy due to androgen receptor variants that promote aberrant activation and neoplastic progression. In recent years, scientific research has focused on the development and identification of natural compound–based drugs. Many natural compounds appear to be effective in enhancing the therapeutic effect of common chemotherapeutic drugs and could be used in combination therapy, reducing side effects and overcoming drug resistance mechanisms. Among natural molecules, S-adenosyl-L-methionine (AdoMet or SAM) has been extensively studied as a therapeutic compound, and its application, alone or in combination with other molecules, is emerging as a potential effective strategy for cancer treatment and chemoprevention. AdoMet is a sulfur-containing nucleoside ubiquitous in all living organisms that plays an indispensable role in cellular metabolism, participating in fundamental biochemical reactions, the most relevant being transmethylation, transsulfuration, and polyamine biosynthesis. The research carried out during my PhD program is part of a broader project aimed at developing new and alternative therapeutic approaches for the treatment of prostate cancer, with the goal of addressing the increased mortality of patients affected by this aggressive tumor. During the three years of my PhD, I evaluated the antiproliferative effects of AdoMet on mCRPC cells and the potential of this sulfonium compound to overcome multidrug resistance mechanisms induced by treatment with cabazitaxel (CBZ), one of the most commonly used chemotherapeutic drugs in clinical practice for prostate cancer treatment. Recent findings have highlighted the role of AdoMet as a modulator of a wide variety of signaling pathways involved in carcinogenesis, including cell proliferation, apoptosis, and autophagy. It is important to note that AdoMet is an FDA-approved dietary supplement, which makes it suitable for therapeutic purposes without the common contraindications of chemotherapeutic drugs. To assess the antiproliferative effects of AdoMet, cell viability assays were first performed after treatment with increasing doses of AdoMet (from 62 μM to 1 mM) for different time points (24, 48, and 72 hr) using two PCa cell lines, PC-3 and DU 145. The results showed that AdoMet exerts an inhibitory effect on cell proliferation in a dose- and time-dependent manner, inducing a 50% reduction in cell viability after 72 hr at a concentration of 400 μM. Subsequently, to evaluate the chemosensitizing effects of AdoMet on the antiproliferative activity of CBZ, cell viability assays were performed after 72 hr of treatment with increasing doses of CBZ, alone or in combination with 400 μM AdoMet. The results suggest that mCRPC cells are more sensitive to CBZ treatment when combined with AdoMet, confirming that AdoMet can enhance the cytotoxic effect of CBZ. Glutathione (GSH) is a key component of the cellular antioxidant system. In cancer cells, elevated GSH levels neutralize reactive oxygen species (ROS) and detoxify xenobiotics. Depletion of intracellular GSH makes neoplastic cells more vulnerable to oxidative stress and chemotherapeutics. Analysis of GSH levels after treatment with 400 μM AdoMet and 0.7 nM or 1.5 nM CBZ, administered individually or in combination for 48 and 72 hr in DU 145 and PC-3 cell lines, revealed a significant reduction of GSH in cells treated with the combination therapy compared to controls and single-agent treatments. This effect was more pronounced after 72 hr, especially in DU 145 cells. The substantial decrease in GSH suggests that the combined treatment effectively compromises the antioxidant defenses of mCRPC cells, increasing their sensitivity to oxidative damage. Supporting this, Western blot analysis showed that AdoMet, CBZ, and especially their combination reduced the expression levels of key antioxidant enzymes involved in maintaining redox homeostasis and protecting against ROS- induced damage, such as GPX4 and catalase. Compounds that increase ROS represent a promising therapeutic strategy, as intensifying oxidative stress in cancer cells, combined with GSH depletion, enhances their susceptibility to chemotherapy. Intracellular ROS levels were also measured by flow cytometry using a CellROX assay, a weakly fluorescent dye that, upon ROS oxidation, emits a strong and stable green fluorescence. In DU 145 cells, single treatments did not alter ROS levels, while the combination of AdoMet and CBZ led to a 1.6-fold increase. In PC-3 cells, AdoMet induced a slight increase and CBZ a more marked rise, but the combined therapy produced the most significant effect, with a 2.4-fold increase compared to control. Furthermore, to evaluate whether high ROS levels caused DNA damage in mCRPC cells, the expression of γH2AX, the phosphorylated form of histone H2AX and a sensitive marker of genomic damage, was analyzed. Immunofluorescence and Western blot showed increased γH2AX signal intensity in treated nuclei, particularly in the combination treatment, highlighting the synergistic interaction between the drug and the sulfonium compound in causing DNA damage. The results obtained led me to investigate morphological alterations in mCRPC cells after treatment with AdoMet and/or CBZ, to study the effects of the sulfonium compound on cell death by mitotic catastrophe, a mechanism considered tumor-suppressive as it directs cells unable to complete mitosis toward death. Mitotic catastrophe is an aberrant form of mitosis characterized mainly by misassembled spindles and abnormal chromosome arrangements. These morphological features were used to study this phenomenon. Specifically, immunofluorescence was performed using an α-tubulin antibody to visualize the microtubule network and mitotic spindle, while chromosomes were stained with Hoechst 33258. Images revealed well-organized mitotic spindles and properly aligned chromosomes on the metaphase plate in control cells, whereas AdoMet- and CBZ-treated cells showed disorganized chromosomes and absence of a mitotic spindle. This spindle disorganization was even more evident when AdoMet and CBZ were administered in combination. To further investigate the antiproliferative effects of AdoMet, CBZ, and their combination in DU 145 and PC-3 cell lines, the main mechanism of cell death, apoptosis, was assessed by FACS analysis. After 72 hr, a significant increase in apoptosis was observed in cells treated with the combination compared to single treatments. These FACS results were confirmed by Western blot analysis of cleaved poly ADP-ribose polymerase (PARP), which was particularly evident in the combination treatment, confirming the synergistic activation of apoptosis by AdoMet and CBZ. In conclusion, these results confirm that AdoMet exerts pleiotropic effects in promoting tumor cell death by modulating ROS and spindle integrity and suggest the possible use of this important and versatile physiological compound, in combination with CBZ, for the development of low-cost therapeutic strategies targeting prostate cancer patients. AdoMet is one of the most studied epigenetic regulators and a known modulator of miRNAs in many biological processes, including cancer progression. To gain new insights into the molecular mechanisms underlying AdoMet’s antitumor activity and to evaluate whether AdoMet can act as an epigenetic regulator of miRNAs in mCRPC cells, we analyzed the expression profile of miRNAs involved in PCa pathogenesis. To this end, quantitative Real- Time PCR (qRT-PCR) experiments were performed after treatment with 400 μM AdoMet for 72 hr in DU 145 cell line. The analysis showed that AdoMet treatment was able to modulate the expression levels of miR-34a and miR-21. MiR-34a is a tumor suppressor involved in regulating pro-apoptotic genes and cell cycle control. Conversely, miR-21, often upregulated in prostate cancer, promotes tumor progression by inhibiting tumor suppressor genes such as PTEN, contributing to increased cellular aggressiveness and treatment resistance. To broaden the study of miRNA regulation after AdoMet treatment, an exploratory bioinformatic analysis was performed. In particular, edgeR, an R-based software for RNA-seq data analysis that applies negative binomial distribution models to estimate variability and detect differential gene expression, was used. Enrichment analyses through GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) were then conducted to interpret the biological pathways involved. These preliminary analyses form the basis for a more in-depth interpretation of the data, allowing hypotheses on the potential pathways and biological processes regulated by AdoMet. Further studies will be required to validate and clarify the functional significance of these findings.