Autophagy and oxidative stress modulation mediate Bortezomib resistance in prostate cancer.

Proteasome inhibitors such as Bortezomib represent an established type of targeted treatment for several types of hematological malignancies, including multiple myeloma, Waldenstrom's macroglobulinemia, and mantle cell lymphoma, based on the cancer cell's susceptibility to impairment of the proteasome-ubiquitin system. However, a major problem limiting their efficacy is the emergence of resistance. Their application to solid tumors is currently being studied, while simultaneously, a wide spectrum of hematological cancers, such as Myelodysplastic Syndromes show minimal or no response to Bortezomib treatment. In this study, we utilize the prostate cancer cell line DU-145 to establish a model of Bortezomib resistance, studying the underlying mechanisms. Evaluating the resulting resistant cell line, we observed restoration of proteasome chymotrypsin-like activity, regardless of drug presence, an induction of pro-survival pathways, and the substitution of the Ubiquitin-Proteasome System role in proteostasis by induction of autophagy. Finally, an estimation of the oxidative condition of the cells indicated that the resistant clones reduce the generation of reactive oxygen species induced by Bortezomib to levels even lower than those induced in non-resistant cells. Our findings highlight the role of autophagy and oxidative stress regulation in Bortezomib resistance and elucidate key proteins of signaling pathways as potential pharmaceutical targets, which could increase the efficiency of proteasome-targeting therapies, thus expanding the group of molecular targets for neoplastic disorders

Effects of Bortezomib on main cell cycle regulators and signaling pathways.

(A) Western blot analyses of key cell cycle proteins p21, p27, and p53 and the proliferation marker PCNA. The experiments were conducted after 24 h of Bortezomib incubation, following a 48 h Bortezomib deprivation of RB60 cells. The + corresponds to the low dose of 20 nM Bortezomib, and the ++ corresponds to the medium dose of 60 nM. The cells were lysed using ice-cold lysis buffer, and protein concentrations were determined using the Bradford assay. The same amounts of total protein were loaded on 12% SDS-PAGE gels, and the transfer was performed using the semidry system. (B) Immunocytochemical staining of naïve DU-145 cells with antibodies against p21 and DAPI to visualize DNA content to assess the localization of p21. Following treatment with 20 nM Bortezomib, the nuclear localization of p21 was verified, indicating the pro-apoptotic role of p21 rather than its pro-survival cytosolic presence. (C) The main pro-survival and proliferation pathways were assessed following the incubation of cells with Bortezomib. All experiments were conducted after 24 h of Bortezomib incubation following a 48-hour drug deprivation from the resistant clones, and the protein quantity was confirmed using the Bradford assay.</p

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Proteasome inhibitors such as Bortezomib represent an established type of targeted treatment for several types of hematological malignancies, including multiple myeloma, Waldenstrom’s macroglobulinemia, and mantle cell lymphoma, based on the cancer cell’s susceptibility to impairment of the proteasome-ubiquitin system. However, a major problem limiting their efficacy is the emergence of resistance. Their application to solid tumors is currently being studied, while simultaneously, a wide spectrum of hematological cancers, such as Myelodysplastic Syndromes show minimal or no response to Bortezomib treatment. In this study, we utilize the prostate cancer cell line DU-145 to establish a model of Bortezomib resistance, studying the underlying mechanisms. Evaluating the resulting resistant cell line, we observed restoration of proteasome chymotrypsin-like activity, regardless of drug presence, an induction of pro-survival pathways, and the substitution of the Ubiquitin-Proteasome System role in proteostasis by induction of autophagy. Finally, an estimation of the oxidative condition of the cells indicated that the resistant clones reduce the generation of reactive oxygen species induced by Bortezomib to levels even lower than those induced in non-resistant cells. Our findings highlight the role of autophagy and oxidative stress regulation in Bortezomib resistance and elucidate key proteins of signaling pathways as potential pharmaceutical targets, which could increase the efficiency of proteasome-targeting therapies, thus expanding the group of molecular targets for neoplastic disorders.</div

List of primary antibodies used for western blot analyses.

List of primary antibodies used for western blot analyses.</p

Ubiquitin-Proteasome system assessment in naïve DU-145, DU-145 RB60U, and DU-145 RB60 resistant cells.

We examined the function of the UPS with Western blots of polyubiquitinated proteins. (A) An initial dose-response experiment was conducted on all three clones (naïve DU-145, DU-145 RB60U, and DU-145 RB60) following 24 h of incubation with Bortezomib. (B) Time-course experiments verified the stable ubiquitination levels at the key intervals of 24, 36, and 48 h following incubation with 20 nM Bortezomib, validating the 24-hour time point as an adequate time point to study effects on main signaling pathways. (C) Further dose-response experiments verified the susceptibility of DU-145 RB60 cells to Bortezomib at concentrations greater than 180 nM. (D) PSMB5 is the most ubiquitous proteasome subunit exhibiting chymotrypsin-like (ChT-L) activity. Following treatment with Bortezomib, we used Western blot analysis to estimate the PSMB5 protein levels. (E) The results were quantified using ImageJ, and to compare the groups, two-tailed t-tests were performed in GraphPad Prism 8. Between naïve and resistant cells, statistically significant differences were documented, regarding PSMB5 accumulation (PF) We estimated the ChT-L activity of naïve DU-145 and DU-145 RB60 cells using fluorometry. The resistant cells exhibited almost 3-fold increased proteasome activity compared to the naïve clone (PA-E) were performed in triplicate, and wherever bar charts are shown; the bars represent the means, and the error bars are the SEM.</p

IC₅₀ Calculation of naïve DU-145, DU-145 RB60, and DU-145 RB60U cells.

The data from crystal violet assays were analyzed using GraphPad Prism 8, and the calculated IC50 values are presented here. The resistant cells exhibited a 5-fold increase in Bortezomib tolerance after 24 weeks of Bortezomib presence, which was augmented more following another 24 weeks of drug presence, reaching a more than 10-fold increase compared to the naïve clone. Additionally, to some extent, cross-resistance to Carfilzomib was observed; the DU-145 RB60 clone achieved 4-fold Carfilzomib resistance compared to the naïve clone after 24 weeks and an almost 6-fold change after another 24 weeks. The long-deprived clone maintained its acquired resistance to both inhibitors during the 24-week monitoring. Regarding resistance to doxorubicin, all three cell clones tested exhibited a similar response to doxorubicin, regardless of resistance to Bortezomib. The experiments were repeated in triplicate, and for the IC50 calculation, the built-in model from GraphPad Prism 8 was used.</p

Wound healing assay of DU-145 naïve and DU-145 RB60 cells.

Cells were seeded on 6-well plates and left to form monolayers. After reaching the desired confluency, wounds were scratched, and the Bortezomib-free media were replaced with medium containing 10% FBS and Bortezomib. The naïve cells were assessed using 20 nM of Bortezomib, and the DU-145 RB60 cells were assessed under the influence of 60 nM Bortezomib. Compared to the control group, the DU-145 naïve cells’ ability to heal wounds was heavily impaired by Bortezomib, while the same effect was not observed on the DU-145 RB60 cells. The resistant cells were able to completely heal the scratches after 72 h of incubation, and the same was achieved by the untreated naïve cells. (TIF)</p

Autophagy and oxidative stress assays using western blots, flow cytometry and confocal microscopy.

(A) Western blot analyses of key proteins regulating autophagy (LC3, Beclin-1, p62) and stress markers (Hsp70 and phosphor-p38). The experiments were conducted after 24 h of Bortezomib incubation following a 48 h Bortezomib deprivation of RB60 cells, as previously noted. The + corresponds to the low dose of 20 nM Bortezomib, and the ++ corresponds to the medium dose of 60 nM. (B) Staining of naïve DU-145 cells with Lysotracker RED, which stains acidic proteins, following treatment with 20 nM Bortezomib. The cells were cultured on coverslips and stained (without fixation) with Lysotracker RED for 15 min at 37°C, followed by confocal imaging. (C) Flow cytometry analysis of Lysotracker RED inside naïve and resistant live cells. The cells were trypsinized and subsequently stained with Lysotracker RED for 45 min followed by analysis using a FACS Calibur flow cytometer. (D) Flow cytometry analysis of ROS generation using H2DCFDA. The cells were incubated for 24 h with Bortezomib and then trypsinized. During the staining procedure, they were maintained inside the culture medium to avoid heat shock and starvation stress. Staining was performed at 37°C and the samples were rinsed with PBS and analyzed using a FACS Calibur flow cytometer.</p

Poly-Unsaturated Fatty Acids (PUFAs) from <i>Cunninghamella elegans</i> Grown on Glycerol Induce Cell Death and Increase Intracellular Reactive Oxygen Species

Cunninghamella elegans NRRL-1393 is an oleaginous fungus able to synthesize and accumulate unsaturated fatty acids, amongst which the bioactive gamma-linolenic acid (GLA) has potential anti-cancer activities. C. elegans was cultured in shake-flask nitrogen-limited media with either glycerol or glucose (both at ≈60 g/L) employed as the sole substrate. The assimilation rate of both substrates was similar, as the total biomass production reached 13.0–13.5 g/L, c. 350 h after inoculation (for both instances, c. 27–29 g/L of substrate were consumed). Lipid production was slightly higher on glycerol-based media, compared to the growth on glucose (≈8.4 g/L vs. ≈7.0 g/L). Lipids from C. elegans grown on glycerol, containing c. 9.5% w/w of GLA, were transformed into fatty acid lithium salts (FALS), and their effects were assessed on both human normal and cancerous cell lines. The FALS exhibited cytotoxic effects within a 48 h interval with an IC50 of about 60 μg/mL. Additionally, a suppression of migration was shown, as a significant elevation of oxidative stress levels, and the induction of cell death. Elementary differences between normal and cancer cells were not shown, indicating a generic mode of action; however, oxidative stress level augmentation may increase susceptibility to anticancer drugs, improving chemotherapy effectiveness

Chemotactic assay of DU-145 naive and DU-145 RB60 cells using Boyden chambers.

Cells were transferred into a chamber containing serum-free medium with or without Bortezomib. The chambers were placed inside microplates’ wells containing medium supplemented with 20% FBS and left to migrate for 24 h. The DU-145 cells, when exposed to Bortezomib (20 nM), decreased their migration rate. Inhibition of migration was also observed when Bortezomib was added to the lower compartment, indicating a chemorepellent role. The DU-145 RB60 cells were also repelled by Bortezomib (60 nM), while the presence of the drug in the upper compartment induced migration towards the other side of the membrane, where Bortezomib was absent. (TIF)</p