Pristimerin Suppresses Janus Kinase/Signal Transducers and Activators of Transcription Oncogenic Signaling To Drive Mitochondrial and Caspase-Dependent Apoptosis in Multiple Myeloma

Abstract

Multiple myeloma (MM) is a plasma cell malignancy characterized by uncontrolled clonal expansion within the bone marrow, and despite major therapeutic advances, it remains largely incurable. The identification of novel agents capable of overcoming apoptosis resistance and targeting critical survival pathways is therefore essential. This study explored in vitro the anticancer potential of pristimerin (Prist), a natural quinone methide triterpenoid, in MM. Prist significantly inhibited the viability of U266 and RPMI8226 cells in a dose-dependent manner and induced cell cycle arrest in the SubG0/G1 phase, indicating apoptotic cell death. Mechanistic studies revealed that Prist triggers apoptosis via caspase cascade activation and targets the JAK/STAT signaling pathway, a critical oncogenic driver in MM. Network pharmacology and molecular docking suggests Prist’s plausible binding with the STAT3 active site, a key component of the JAK/STAT pathway, supporting its inhibitory role. Western blot analysis demonstrated that Prist reduced the constitutive phosphorylated STAT3 (p-STAT3) as well as IL6- IL6-stimulated p-STAT3 levels in MM cells, further validating STAT3 pathway inactivation. Importantly, Prist synergistically enhanced the cytotoxic effects of bortezomib, further amplifying caspase activation and apoptotic signaling. Collectively, these findings identify Prist as a potent natural compound that suppresses MM progression by inducing apoptosis and blocking STAT3-driven oncogenic signaling. This study highlights Prist’s therapeutic promise and supports its further evaluation as a standalone or combination strategy for MM treatment.

Multiple myeloma (MM) is a malignant plasma cell disorder characterized by the clonal expansion of abnormal B-lymphocyte-derived plasma cells in the bone marrow, leading to excessive monoclonal immunoglobulin production and progressive end-organ dysfunction. Although major therapeutic advances, including immunomodulatory drugs (IMiDs), proteasome inhibitors, monoclonal antibodies, corticosteroids, and autologous hematopoietic stem cell transplantation, have significantly improved patient outcomes, MM remains largely incurable. Most patients eventually relapse due to the persistence of drug-resistant clones and the supportive influence of the tumor microenvironment. Despite modern treatment regimens, the five-year survival rate remains suboptimal at approximately 40–49%, with marked disparities driven by biological heterogeneity, treatment accessibility, and comorbidities. Therefore, finding new therapeutic strategies to target survival signaling networks and overcome drug resistance mechanisms is of considerable clinical importance.

One of the key features of MM biology is the activation of multiple oncogenic pathways that promote survival, proliferation, immune evasion, and resistance to apoptosis. Among these, the JAK/STAT3 pathway is especially important. Persistent STAT3 activation is observed in most MM cases and is driven by both internal mutations and external cytokines, particularly interleukin-6 (IL-6) produced by the bone marrow microenvironment. Constitutively active STAT3 controls the expression of many antiapoptotic and pro-survival genes, including Bcl-2, Bcl-xL, Mcl-1, survivin, and cyclin D1, thus supporting MM progression and resistance to therapy. , Targeting STAT3 signaling is therefore seen as a promising therapeutic approach to overcoming apoptosis resistance and enhancing clinical response in MM.

Natural compounds have become appealing options for cancer treatment because of their structural variety, multitargeted mechanisms, and lower toxicity compared to traditional chemotherapies. Among these, Pristimerin (Prist), a natural quinone methide triterpenoid extracted from plants of the Hippocrateaceae and Celastraceae families, has gained notable interest. Chemically, it is known as 20α-3-hydroxy-2-oxo-24-nor-friedela-1,10,3,5,7-tetraen-carboxylic acid-29-methylester, and Prist has shown strong anticancer effects in various tumor models. Earlier studies reveal that Prist inhibits tumor cell growth, prevents metastasis and blood vessel formation, and promotes apoptosis and autophagy in several cancers, including breast, lung, prostate, and blood-related malignancies. − Mechanistically, Prist is reported to affect key molecular pathways, including NF-kB, proteasome activity, MAPK signaling, Noxa-dependent apoptosis via activation of the FoxO3a pathway in esophageal squamous cell carcinoma and mitochondrial-induced cell death, suggesting a broad range of antitumor effects. ,

Given the central role of STAT3 in MM pathogenesis and the increasing evidence supporting Prist’s anticancer activity, we aimed to determine whether Prist could effectively target survival pathways in MM. In this study, we systematically assessed the antiproliferative and pro-apoptotic effects of Prist across various MM cell lines. We also used molecular docking and network pharmacology to identify potential interactions between Prist and its targets, with a particular focus on the JAK/STAT3 axis. By combining biochemical, molecular, and computational methods, our research offers new insights into the therapeutic potential of Prist in MM and highlights STAT3 inhibition as a key mechanism behind its cytotoxic effects. These results emphasize the importance of phytochemical-based therapies in overcoming apoptosis resistance and enhancing future treatment options for MM.

Materials and Methods

Reagents and Antibodies

RPMI-1640, FBS, and PenStrep were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Sigma-Aldrich supplied the Cell Counting Kit-8 (CCK-8) (catalog number:96992), Prist­(catalog number: P0020), and N-acetylcysteine (catalog number: A9165) . The live/dead cell viability/cytotoxicity kit (catalog number:L3224A,B) was obtained from Molecular Probes (Eugene, Oregon, USA). The Annexin-V FITC apoptosis detection kit, fixation/permeabilization solution kit, JC-1, BV421 mouse antigamma H2AX (pS139), PE rabbit antiactive caspase-3, and Alexa Fluor 700 mouse anticleaved PARP (Asp214) were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Antibodies against p-H2AX (catalog number: 2577S), cytochrome c (catalog number:11940), caspase-3 (catalog number:9668S), cleaved caspase-3 (catalog number:9664S), caspase-9 (catalog number:9508S), PARP (catalog number:9542L), Bcl-2­(catalog number:15071S), Bax (catalog number:2774), Bcl-xL­(catalog number:2762S), BID (catalog number: 2002S), XIAP­(catalog number: 2042L), p21 (catalog number: 2947S), p27 (catalog number: 3686), cyclin D1­(catalog number: 2922), STAT3 (catalog number: 9132), and p-STAT3 (catalog number:94994S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Loading control antibodies, including GAPDH (catalog number: 32233) and HSP60 (catalog number: 13115), were sourced from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies were diluted as per the concentration mentioned in manufacturer’s protocol.

Cell Culture

U266, MM.1S, and RPMI8226 MM cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and cultured in RPMI-1640 medium supplemented with 20% (v/v) fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin (PenStrep) at 37 °C in a humidified incubator with 5% CO₂.

Cell Viability Assay

The antiproliferative activity of Prist on MM cell lines was evaluated using CCK-8. A total of 1 × 10⁴ cells/well were seeded in a 96-well plate with varied Prist concentrations (0.1–4 μM) for 24 h. After incubation, 10 μL of CCK-8 was added to each well, then the plate was incubated at 37 °C. The optical density (OD, absorbance) was determined at 450 nm using a Tecan microplate reader (Tecan BioTek Instruments Inc.). The percentage of viable cells was calculated as described previously.

Live/Dead Cell Assay

U266, MM.1S, and RPMI8226 cells were treated with increasing concentrations of Prist and then incubated for 24 h. The live/dead stain consisted of 5 μL of ethidium homodimer-1 (EthD-1) and 5 μL of calcein AM added to 10 mL of PBS (final concentrations of 1 μM EthD-1 and 2 μM calcein). The treated cells were incubated with this stain solution for 30 min in the dark, then cell images were taken at 20× magnification using the EVOS FLoid cell imaging system (Thermo Fisher Scientific).

Measurement of Apoptosis

The Annexin V/propidium iodide (PI) dual staining assay was used to study Prist-induced apoptosis.After being treated with curcumin for 24 h, U266 and RPMI cells were centrifuged, cleaned with ice-cold PBS, and then stained for 20 min with Annexin V-FITC and PI in 1× binding buffer. The cells were categorized as necrotic, early apoptotic (Annexin FITC positive, PI negative), late apoptotic (Annexin FITC positive, PI positive), or alive (Annexin FITC negative, PI negative) using the BD LSR Fortessa analyzer (BD Biosciences). The percentages of early and late apoptotic cells were combined to get the percentage of apoptotic cells. The total percentage of apoptotic cells was calculated as the sum of early and late apoptotic cells percentages.

Cell Cycle Analysis

U266 and RPMI8226 cells were treated with Prist for 24 h. The cells were stained with PI for 30 min at 37 °C. The cell cycle distribution was examined using flow cytometry with the BD LSRFortessa analyzer (BD Biosciences) as described previously.

Target Identification and Molecular Docking of Prist with STAT3

The canonical SMILES structure of Prist was retrieved from PubChem and uploaded independently to three target prediction platforms: SwissTargetPrediction, Super-PRED, and Way2Drug. , For SwissTargetPrediction, only targets with a probability score ≥ 0.1 were retained. From Super-PRED and Way2Drug, predicted targets ranked within the top confidence categories were selected. After removing duplicates, all predicted protein targets were standardized to official gene symbols using UniProt. Multiple myeloma-related genes were collected from the GeneCards database by searching the keyword "Multiple myeloma". Genes with a relevance score above the median value were retained to ensure biological significance. The intersecting genes between Prist-predicted targets and disease-associated genes were identified using Venn analysis and considered potential therapeutic targets. These overlapping targets were imported into the STRING database to construct the protein–protein interaction (PPI) network, restricting the organism to Homo sapiens and applying the highest confidence interaction score (≥0.900). Disconnected nodes were removed. The resulting PPI network was visualized and analyzed in Cytoscape v3.8.2. Hub genes were identified using the CytoHubba plugin based on the degree algorithm, selecting the top-ranked genes as core targets. For molecular modeling, the crystal structure of STAT3 (PDB ID: 6TLC) was obtained from the Protein Data Bank Protein preparation included removal of crystallographic water molecules, addition of polar hydrogens, assignment of Kollman charges, and energy minimization. Prist was geometry-optimized prior to docking. Induced-fit docking was performed using AutoDockFR, allowing flexibility of key active-site residues to better capture ligand-induced conformational changes. The docking grid was centered on the STAT3 binding pocket reported in the crystal structure. Multiple docking poses were generated, and the best complex was selected based on binding energy and interaction profile. Protein-ligand interactions were analyzed using PyMOL and Schrödinger Maestro (academic version).

Immunoblotting Technique

U266 and RPMI8226 cells were collected after 24 h of Prist treatment. Proteins were separated using SDS-PAGE and then moved to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). Proteins were measured with a NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA), and then β-mercaptoethanol was added. After cell lysates were prepared, 30–50 μg of protein were added, separated on SDS-PAGE, and then transferred to a PVDF membrane (Bio-Rad). Protein concentrations were then quantified using the ND-1000 spectrophotometer (Nanodrop Technologies, Thermo Scientific). Multiple antibodies were used for immunoblotting, and a ChemiDoc system (Bio-Rad) was used for visualization. Western blot experiments for only key proteins were performed in triplicate.

Reactive Oxygen Species and Mitochondrial Superoxide Determination

Post Prist treatment for 24hours, multiple myeloma cells were washed with Hank’s Balanced Salt Solution (HBSS) and stained for 20 min at 37 °C with 5 μM CellROXTM Green Reagent (Invitrogen, MA, USA) for determination of ROS at cellular level or with the MitoSOX Red Mitochondrial Superoxide Indicator (Invitrogen, MA, USA) in HBSS for determination of ROS at mitochondrial level. Cells were then subjected to flow cytometry (Ex: 488, Em: 575/26) to assess cellular/mitochondrial superoxide levels, a biomarker of oxidative stress.

Flow Cytometry Analysis of Active Caspase 3 and Cleaved PARP

Following 24 h of Prist treatment (0, 0.25, 0.5, and 1 μM), cells were fixed and permeabilized using the BD Cytofix/Cytoperm Plus Fixation and Permeabilization kit. Antibodies against Caspase-3-BV605 and PARP Cleaved Form-AF700 were used to stain 0.3 × 10⁶ cells in HBSS for 30 min. After another rinse with HBSS, the cells were resuspended in the same solution. The percentage of cells containing active caspase-3 and cleaved PARP was evaluated using flow cytometry on a BD LSRFortessa analyzer (BD Biosciences).

Measurement of DNA Double-Strand Breaks

MM cells were treated with Prist at various concentrations for 24 h. After incubation, the cells were fixed and permeabilized with a BD Cytofix/Cytoperm Plus Fixation and Permeabilization Kit according to the manufacturer’s directions. The H2AX (pS139)–Alexa Fluor 647 antibody was used to stain 0.3 × 10⁶ cells in HBSS for 30 min at room temperature. Following labeling, DNA damage was detected and quantified using flow cytometry using a BD LSRFortessa analyzer (BD Biosciences).

Measurement of Mitochondrial Membrane Potential (MMP)

U266 and RPMI8226 cells were treated with Prist at varied doses for 24 h. The cells were stained with membrane-permanent JC-1, and the mitochondrial membrane potential was assessed using flow cytometry on a BD LSRFortessa analyzer (BD Biosciences).

Generation of EBV-B Lymphoblastoid Cell Lines

In vitro transformation of B lymphocytes using Epstein–Barr Virus (EBV) concentrated supernatant is a routine procedure for obtaining immortalized human Lymphoblastoid B cell lines (B-LCL) for use in preclinical research studies. We generated B-LCLs in our laboratory by infecting a healthy donor’s B cells with EBV-containing supernatant from B95–8 cell culture, commercialized by ATCC as VR-1492. Briefly, we isolated Peripheral Blood Mononuclear Cells (PBMCs) from a healthy individual’s peripheral blood using Ficoll Paque Premium density gradient (GE Healthcare) and SepMate tubes (STEMCELL Technologies) according to the manufacturer’s instructions. These PBMCs were cryopreserved in 10% DMSO + FBS and kept in liquid nitrogen until further use. The blood sample was obtained from a healthy volunteer donor who signed an IRB-approved written informed consent under the research project IRGC-07-SI-20-716.

On the day of the experiment, one vial of cryopreserved PBMCs was thawed in 10 mL RPMI media complemented with 10% FBS. The cells were centrifuged, counted, and then 3 × 10⁶ cells were collected for the assay. The cells were washed by centrifugation, and the pellet was resuspended in 1 mL of freshly thawed EBV supernatant (ATCC VR-1492). Further, the cell suspension was incubated in a water bath at 37 °C for 1 h with rigorous agitation every 10 min. Further, 3 mL of transformation medium was added (Advanced RPMI 1640 + 20% FBS + 200 ng/mL cyclosporine), and the cells were incubated at 37 °C, 5% CO₂, in a standing T-25 flask. One week after culture initiation, we observed the cells for signs of transformation, including spike-like projections and formation of cell clusters. The cells were cultured for at least 2–3 weeks before use in flow cytometry and proliferation assays.

Statistical Analysis

To compare the groups, we used a one-way ANOVA and Tukey’s multiple comparison test. The statistical analysis was performed using GraphPad Prism (version 5.0 for Windows; GraphPad Software Inc., http://www.graphpad.com). The data is presented as mean ± standard deviation. Statistical significance is shown by values of *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001, respectively.

Results

Prist Suppresses Cell Viability and Induces Apoptosis in MM Cells

We first investigated the antiproliferative effects of Prist across multiple myeloma (MM) cell lines. Treatment of U266, RPMI8226, and MM.1S cells with increasing Prist concentrations (0–4 μM for 24 h) resulted in a considerable cell growth inhibition as evident from IC₅₀ (0.162 μM and 0.310 μM for U266 and RPMI 8226 respectively) and from Figure A. Consistently, Prist induced pronounced morphological changes characteristic of cytotoxicity in all tested cell lines (Figure S1). Live/dead staining further confirmed these findings, showing a dose-dependent increase in EthD-1-positive (dead) cells following Prist exposure (Figure B).

1.

Prist reduces cell viability and induces cell-cycle arrest and apoptosis in multiple myeloma (MM) cells.(A) U266, RPMI8226, and MM.1S cells were treated with increasing concentrations of Prist (0, 0.10, 0.25, 0.75, 1, 2, and 4 μM) for 24 h. Cell viability was assessed using the CCK-8 assay as described in Materials and Methods. Data represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Prist induces cell death in MM cell lines. U266, RPMI8226, and MM.1S cells were treated with 0.25, 0.5, or 1 μM Prist for 24 h, stained with a live/dead viability reagent, and imaged using the EVOS FLc Cell Imaging System (Invitrogen, Thermo Fisher Scientific) at 20× magnification (scale bar = 200 μm). (C, D) Prist alters cell-cycle distribution and promotes accumulation of apoptotic (sub-G0/G1) populations. (C) U266 and (D) RPMI8226 cells were treated with 0, 0.25, 0.5, or 1 μM Prist for 24 h, stained with propidium iodide (PI), and analyzed for DNA content by flow cytometry. Graphs show mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Prist downregulates key cell-cycle regulatory proteins. U266 and RPMI8226 cells were treated with 0, 0.25, 0.5, or 1 μM Prist for 24 h. Whole-cell lysates were subjected to Western blotting using antibodies against CDK4, CDK6, cyclin B1, and HSP60.

To determine whether growth inhibition was associated with cell-cycle dysregulation, we examined DNA content profiles in U266 and RPMI8226 cells. Prist treatment significantly increased the sub-G0/G1 population in a concentration-dependent manner (Figure S1 and Figure C,D), indicating apoptotic DNA fragmentation. This was accompanied by a reduction in essential cell-cycle regulators, including cyclin D1, cyclin B1, CDK4, and CDK6 (Figure E), supporting Prist’s role in enforcing cell-cycle arrest.

We next evaluated whether Prist-induced growth inhibition occurs primarily via apoptosis. Annexin V/PI staining demonstrated a significant, dose-dependent increase in apoptotic cells in both U266 and RPMI8226 following Prist treatment (Figure A–C). Moreover, Prist markedly increased p-H2AX levels (Figure S1), indicating DNA double-strand breaks, a hallmark of apoptotic initiation.

2.

Prist induces apoptosis in MM cells. (A) U266 and RPMI8226 cells were treated with 0, 0.25, 0.5, or 1 μM Prist for 24 h, stained with fluorescein-conjugated annexin V and propidium iodide (PI), and analyzed by flow cytometry to quantify apoptotic populations. (B, C) Quantification of annexin V/PI staining in (B) U266 and (C) RPMI8226 cells. Data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Prist activates the caspase cascade. U266 and RPMI8226 cells treated with the indicated Prist concentrations for 24 h were lysed, and 50 μg of protein per sample was subjected to SDS-PAGE and Western blotting using antibodies against caspase-9, caspase-3, cleaved caspase-3, PARP, and GAPDH. (E–H)­Densitometric analysis of the key genes involved in caspase cascade. Caspase 3 (E) U266 and (F) RPMI8226 and cleaved caspase 3 (G) U266 and (H) RPMI8226. Data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Flow cytometric analysis of PARP cleavage in (I) U266 and (J) RPMI 8226 following Prist treatment, as described in Materials and Methods. Data represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To delineate downstream apoptotic signaling events, we assessed caspase activation. Immunoblot analysis revealed dose-dependent activation of caspase-9, caspase-3, and PARP cleavage in Prist-treated MM cells (Figure D). Densitometric analysis revealed significant downregulation of caspase-3 with upregulation of cleaved caspase 3 as seen in Figure E–H. Flow cytometry quantification corroborated the induction of cleaved PARP (Figure I,J andFigure S2). Collectively, these data demonstrate that Prist suppresses MM cell viability primarily through activation of caspase-dependent apoptotic pathways.

Prist Activates Both Intrinsic and Extrinsic Mitochondrial Apoptotic Pathways in MM Cells

To elucidate the molecular mechanisms underlying Prist-induced apoptosis in MM cells, we investigated its effects on key regulators of both the extrinsic (death receptor–mediated) and intrinsic (mitochondrial) apoptotic pathways. Given the central role of mitochondrial integrity in apoptotic commitment, we assessed whether Prist perturbs Bcl-2 family signaling and mitochondrial membrane stability.

Prist treatment led to clear caspase-8 activation in U266 and RPMI8226 cells (Figure A), indicating engagement of the extrinsic pathway. Activated caspase-8 is known to cleave Bid into truncated Bid (tBid), which then translocates to mitochondria to initiate mitochondrial outer membrane permeabilization (MOMP). Consistent with this mechanism, Prist markedly increased Bid truncation (Figure A), establishing a molecular link between death receptor signaling and mitochondrial involvement. The data was supported by the densitometric analysis revealed significant upregulation in the levels of cleaved caspase 8 as seen in both MM cell lines (Figure B,C).

3.

Prist regulates mitochondrial apoptotic signaling pathways in MM cells. U266 and RPMI8226 cells were treated with Prist for 24 h. Cells were lysed and immunoblotted with antibodies against (A) cleaved caspase 8, Bid, and GAPDH. Densitometric analysis of the key genes involved in mitochondrial pathway. Cleaved Caspase 8 (B) U266 and (C) RPMI8226. Data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Western blot data of Bax, Bcl2, and GAPDH. Densitometric analysis of pro-apoptotic protein Bax. Bax (E) U266 and (F) RPMI8226. Data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Prist treatment causes the loss of MMP in MM cells. (G) Post treatment with Prist U266 and RPMI8226 cells were stained and analyzed by flow cytometry for MMP as described in Materials and Methods. The dose-dependent loss of MMP in (H) U266 and (I) RPMI 8226 cells were quantified and represented as a bar graph. The graph displays the mean ±SD of three independent of experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

We next examined canonical regulators of mitochondrial integrity. Prist exposure resulted in a significant upregulation of the pro-apoptotic protein Bax, accompanied by a reduction in the antiapoptotic protein Bcl-2 (Figure D–F).

To directly evaluate mitochondrial function, we assessed mitochondrial membrane potential (MMP) using the JC-1 probe. Prist-treated MM cells exhibited a pronounced loss of MMP in a dose-dependent manner (Figure G–I), reflecting early mitochondrial destabilization. Loss of MMP is a hallmark of intrinsic apoptosis, indicating compromised mitochondrial integrity and initiation of the apoptosome-mediated caspase cascade.

Together, these findings demonstrate that Prist activates a coordinated apoptotic program in MM cells involving both extrinsic caspase-8–mediated signaling and intrinsic mitochondrial disruption. By promoting Bid truncation, altering Bax/Bcl-2 balance, and inducing mitochondrial depolarization, Prist robustly drives mitochondrial apoptotic execution in MM cells.

Target Identification of Prist in MM Cells through a Network Pharmacology Approach and Molecular Docking with STAT3

To investigate the molecular interaction landscape of Prist (Figure A) in the context of MM, we integrated data from several drug–target and disease–gene databases. Using the SMILES of Prist, the Way2Drug database predicted 67 associated targets, while SuperPred identified an additional 119 candidates. SwissTarget Prediction identified an additional 92 potential targets (Figure B). For the disease side, a comprehensive search in the GeneCards database yielded approximately 31,000 genes linked to MM. Intersecting the drug-associated targets with the MM-related genes revealed 127 overlapping targets, representing the most relevant candidates for further exploration.

4.

Identification of Prist targets in MM using network pharmacology and molecular docking. (A) Chemical 2D structure of Prist. (B) Venn diagram showing overlapping genes between Prist-associated targets and MM-related genes. (C) Protein–protein interaction (PPI) network of the 127 overlapping targets. (D) Hub-gene network derived from topological screening of the PPI network. (E) 2D interaction diagram of the Prist-STAT3 complex. (F) 3D molecular docking presentation of Prist binding within the STAT3 active site.

A protein–protein interaction (PPI) network of these 127 shared targets was constructed using the STRING database and subsequently visualized in Cytoscape. The 2D structure of Prist is depicted in Figure A, and the resulting PPI network is shown in Figure C. To pinpoint the most influential nodes in this network, CytoHubba analysis was performed, which highlighted ten hub genes, including STAT3, AKT1, EGFR, MTOR, MMP9, MAPK1, PARP1, KDR, PRKACA, and AR based on the degree score (Figure D). Interestingly, based on the highest scores and biological relevance, STAT3 emerged as the top-ranked hub gene and was selected for further analysis.

STAT3, a transcription factor in the JAK/STAT pathway, is activated by phosphorylation at Tyr705, leading to dimerization, nuclear translocation, and the activation of genes such as survivin, Bcl-xl, and VEGF. In cancers, STAT3 remains persistently active, promoting tumor cell survival. Inhibiting STAT3 signaling induces apoptosis, highlighting its pivotal role in cancer progression. −

To investigate how Prist interacts with STAT3, we employed molecular docking, a key computational approach for understanding drug–target interactions and guiding therapeutic development. The results demonstrated a strong binding affinity, with a docking score of −5.632 kcal/mol and revealed interactions with critical STAT3 active-site residues, including Lys591 and Ser636 (Figure E,F). The literature supports the relevance of these sites, as residues such as Arg609, Glu612, and Ser636 have been identified as pivotal for STAT3 function in breast cancer. At the same time, Lys591, Arg609, Ser611, and Ser636 play similar roles in pancreatic cancer. These overlaps affirm that Prist binds within the established STAT3 active site, providing robust evidence for the specificity and significance of our docking results.

Prist Suppresses Constitutive and IL-6-Driven STAT3 Activation in MM Cells

To experimentally validate the predicted STAT3-targeting potential from network pharmacology and molecular docking analyses, we examined the impact of Prist on STAT3 activation in MM cells. U266 and RPMI8226 cells were treated with increasing concentrations of Prist, and phosphorylation of STAT3 at Tyr705, an essential residue for STAT3 dimerization and transcriptional activity, was evaluated by immunoblotting. Prist treatment resulted in a robust, dose-dependent reduction in constitutive STAT3 phosphorylation without altering total STAT3 protein levels (Figure A), indicating selective inhibition of STAT3 activation rather than global protein downregulation. These findings confirm that Prist directly interferes with STAT3 signaling, consistent with our computational predictions.

5.

Prist inhibits constitutive and IL-6–induced STAT3 and AKT signaling in MM cells. (A) U266 and RPMI8226 cells were treated for 24 h with increasing doses of Prist. Protein lysates were immunoblotted with antibodies against p-STAT3^Tyr705, total STAT3, p-AKT^Ser473, AKT, XIAP, cIAP1, BCL-XL, and GAPDH. Densitometric analysis of STAT3 and AKT post inhibition by Prist. pSTAT3/STAT3 (B) U266 and (C) RPMI8226 and p-AKT/AKT (D) U266 and (E) RPMI8226 Data represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (F) Prist blocks IL-6–induced STAT3 activation. U266 and RPMI8226 cells were pretreated with 1 μM Prist for 24 h, stimulated with IL-6 (100 ng/mL) for 30 min, and analyzed for p-STAT3^Tyr705, STAT3, and GAPDH via Western blotting.

Because STAT3 acts in concert with other pro-survival pathways in MM, we next assessed the effects of Prist on AKT and its downstream antiapoptotic substrates. Prist treatment significantly suppressed AKT phosphorylation at Ser473, accompanied by downregulation of cIAP1, XIAP, and Bcl-XL, key survival proteins that are transcriptionally regulated by STAT3 or AKT signaling (Figure A). Densitometric analysis of the key proteins STAT3 and AKT were found to be significantly downregulated (Figure B–E) by Prist. This coordinated inhibition suggests that Prist disrupts a broader oncogenic signaling network that supports MM cell survival.

IL-6 is a major cytokine in the bone marrow microenvironment that potently activates STAT3 and promotes MM proliferation, survival, and drug resistance. As expected, IL-6 stimulation markedly increased STAT3 phosphorylation in both MM cell lines. However, pretreatment with Prist dramatically attenuated IL-6-induced STAT3 activation (Figure F). This demonstrates that Prist not only inhibits baseline, constitutive STAT3 activity but also effectively blocks extrinsic cytokine-mediated STAT3 stimulation.

Collectively, these results establish Prist as a potent inhibitor of both intrinsic and microenvironment-driven STAT3 signaling in MM cells. By suppressing STAT3 and associated pro-survival pathways, Prist disrupts critical oncogenic circuits required for MM cell viability and resistance.

Prist-Induced Apoptosis Is Critically Dependent on Reactive Oxygen Species (ROS) Generation in MM Cells

Given that elevated reactive oxygen species (ROS) levels represent a well-established mechanism through which many anticancer agents trigger apoptosis we investigated whether ROS generation contributes to Prist-mediated cytotoxicity in MM cells. To assess the impact of Prist on ROS levels, we treated U266 and RPMI 8266 with varying doses of Prist for 24 h, subsequently measuring ROS levels. The flow cytometry data indicated a significant dose-dependent increase in cellular and mitochondrial reactive oxygen species production (Figure S2). We further employed the ROS scavenger N-acetyl-l-cysteine (NAC) to counteract the BTZ-induced ROS production, U266 and RPMI8226 cells were pretreated with N-acetylcysteine (NAC), a potent ROS scavenger, followed by exposure to Prist to determine whether neutralizing ROS could rescue cells from Prist-induced cell death.

Pretreatment with NAC markedly reversed Prist-induced cytotoxic effects, as evidenced by the restoration of normal cell morphology and significant recovery of cell viability (Figure A,B). NAC also prevented Prist-mediated nuclear/DNA damage, consistent with its ability to suppress ROS-driven apoptotic signaling.

6.

Prist-induced ROS generation drives caspase activation and apoptosis in MM cells. U266 and RPMI8226 cells were pretreated with NAC (10 mM) for 30 min, followed by 1 μM Prist for 24 h. (A) Morphological changes observed by light microscopy at 20× magnification. (B) Live/dead staining imaged using EVOS FLc Cell Imaging System (20×; scale bar = 200 μm). (C) Western blot analysis of caspase-3, cleaved caspase-8, PARP, and GAPDH. (D, E) Flow cytometric annexin V/PI analysis of apoptosis in (D) U266 and (E) RPMI 8266. Data represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To further delineate the involvement of ROS in downstream apoptotic cascade activation, we examined key molecular markers of apoptosis. Immunoblot analysis revealed that NAC pretreatment effectively suppressed Prist-induced activation of caspase-3, caspase-8, and cleavage of PARP in both MM cell lines (Figure C), indicating that ROS generation is upstream of caspase activation. Flow cytometry supported these findings: NAC substantially diminished Annexin V/PI–positive apoptotic populations in both U266 and RPMI8226 cells (Figure D,E andFigure S3).

Together, these findings demonstrate that ROS generation is a critical initiating event in Prist-induced apoptosis. By preventing ROS accumulation, NAC abrogates Prist’s ability to induce mitochondrial and caspase-dependent apoptotic signaling, establishing ROS as an essential mediator of Prist’s cytotoxic effects in multiple myeloma cells.

Prist Synergizes with Bortezomib to Potentiate Antitumor Activity in U266 Cells

Bortezomib (BTZ), a frontline proteasome inhibitor for relapsed or refractory multiple myeloma, is limited by the frequent development of drug resistance and subsequent disease relapse. To determine whether Prist could enhance the therapeutic efficacy of BTZ, we evaluated their combined effects on MM cell viability using subtoxic concentrations of each agent.

U266 cells were treated with increasing combinations of Prist and BTZ, and viability was assessed by CCK-8 assay. Drug–drug interactions were quantified using the Chou–Talalay method and CalcuSyn software. A pronounced synergistic effect was observed at 0.25 μM Prist and 5 nM BTZ, yielding a Combination Index (CI) of 0.48066 (Figure A,B andTable ), confirming a strong synergistic interaction (CI < 1). These results indicate that Prist significantly enhances the cytotoxic potential of BTZ even at low, clinically relevant concentrations.

7.

Prist synergizes with bortezomib (BTZ) to enhance antimyeloma activity. U266 cells were treated with various combinations of Prist and BTZ for 24 h. (A, B) In U266 cells, the dose–effect curves for single-agent Prist and BTZ, as generated by CalcuSyn, exhibited sigmoidal plots with high linear correlation coefficients (r = 0.988 for Prist and r = 0.9583 for bortezomib), demonstrating that the Chou–Talalay model accurately represented the experimental data (Figure A and Table ). The combination index (CI) plots indicated that cotreatment with subtoxic concentrations of Prist and BTZ produced CI values below 1 across a broad spectrum of fractional effects (Fa ≈ 0.70–0.85), thereby demonstrating a strong synergistic interaction rather than just additivity (Figure B and Table ). The combination of 0.25 μM Prist and 5 nM BTZ yielded a CI of 0.48066 and significantly enhanced the dose-reduction index (DRI) for both agents, suggesting that comparable cytotoxicity levels can be attained at considerably lower drug concentrations when administered concurrently, potentially mitigating BTZ -related toxicity. (C) Morphological changes (upper panel) and live/dead staining (lower panel) following treatment with subtoxic doses of Prist (0.25 μM), BTZ (5 nM), or the combination. Images acquired at 20× magnification (scale bar = 200 μm). (D) Cell viability measured by CCK-8 following Prist, BTZ, or combination treatment. Data are mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Western blot analysis of p-STAT3, STAT3, caspase-3, cleaved caspase-3, cleaved caspase-8, PARP, and GAPDH.

1. Combination Index Calculation Using Chou and Talalay Method in U266 Cell Lines .

PRIST (μM)	bortezomib (nM)	fractional effect (Fa)	combination index (CI)	concentration reduction index (DRI) PRIST (μM)	concentration reduction index (DRI) bortezomib (nM)
0.1	5	0.706	0.56510	2.20186	9.01363
0.1	10	0.743	0.41887	2.82758	15.3344
0.1	25	0.819	0.20185	5.17147	117.866
0.25	5	0.820	0.48066	2.08752	616.213
0.25	10	0.811	0.52408	1.92581	207.619
0.25	25	0.774	0.75697	1.42114	18.7584
0.5	5	0.844	0.76042	1.31596	1916.26
0.5	10	0.854	0.68467	1.46189	1603.31
0.5	25	0.845	0.75462	1.32954	403.001

^aAbbreviation: PRIST - pristimerin.

Prist Increases BTZ Sensitivity and Augments Apoptotic Signaling

To functionally validate the synergistic interaction, we examined morphological changes and cell death following cotreatment. The combined administration of Prist (0.25 μM) and BTZ (5 nM) markedly reduced U266 cell viability compared to either agent alone, as evidenced by increased cell rounding, loss of adherence, and decreased green fluorescence in live/dead staining (Figure C,D). This confirmed enhanced cytotoxicity under combined treatment conditions.

Next, we investigated whether the enhanced antitumor effect was associated with amplified apoptotic signaling. Immunoblot analysis revealed that dual treatment resulted in a substantial reduction in p-STAT3 and Bid levels, along with a robust increase in cleaved caspase-8, cleaved caspase-3, and PARP cleavage (Figure E). These data suggest that Prist not only disrupts STAT3-mediated survival pathways but also potentiates caspase-dependent apoptosis when combined with BTZ.

Collectively, these findings establish Prist as a potent chemosensitizer that significantly enhances the antimyeloma efficacy of bortezomib through coordinated suppression of STAT3 signaling and amplification of apoptotic cascades. This combination may have important therapeutic implications for overcoming BTZ resistance in MM.

Prist Exhibits Potent Antiproliferative and Proapoptotic Effects in EBV-Transformed B Lymphoblastoid Cell Lines (B-LCLs)

Epstein–Barr Virus (EBV)–driven transformation of B cells is largely mediated by the viral oncoprotein Latent Membrane Protein 1 (LMP1), which constitutively activates major survival pathways, particularly the JAK/STAT axis, to promote uncontrolled proliferation and resistance to apoptosis. To determine whether Prist can counteract these oncogenic signals, we employed EBV-immortalized B lymphoblastoid cell lines (B-LCLs) generated from healthy donor PBMCs, as described in the Materials and Methods. We want to clearly state that B-LCLs are an EBV-transformed B-cell model used to study STAT3-related survival signaling in EBV contexts, rather than a "normal control". The purpose is to test whether Prist’s key mechanistic pathway identified in MMreduction of continuous STAT3 phosphorylation and activation of caspase-dependent apoptosisis also active in an EBV-driven B-cell setting where latent viral programs (notably LMP1) are known to activate survival signaling pathways, including JAK/STAT/STAT3, and provide resistance to cell death

Treatment of B-LCLs with increasing concentrations of Prist (0–1.0 μM) resulted in a clear, dose-dependent suppression of cell viability (Figure A). Morphological examination revealed progressive cytotoxic changes, including cell shrinkage, loss of structural integrity, and increased detachment at higher Prist concentrations (Figure B, upper panel). Consistent with these morphological alterations, live/dead staining demonstrated a concentration-dependent increase in nonviable (EthD-1–positive) cells following Prist exposure (Figure B, lower panel). Quantitative analysis confirmed significant inhibition of viability across nearly all tested doses (Figure C).

8.

Prist suppresses proliferation and induces apoptosis in EBV-immortalized B-lymphoblastoid cells (B-LCLs). (A) B-LCLs were treated with increasing Prist concentrations (0–4 μM) for 24 h, and viability was assessed by CCK-8. Data represent mean ± SD of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Morphological changes (upper panel) and live/dead staining (lower panel) in B-LCLs treated with 0, 0.25, 0.5, or 1 μM Prist for 24 h. (C) Images captured at 20× magnification (scale bar = 200 μm) and number of live cells depicted as bar graph. (D) Annexin V/PI apoptosis analysis following Prist treatment. The graph shows mean ± SD from three independent experiments. (E) Western blot detection of caspase-3, cleaved caspase-3, PARP, and GAPDH in B-LCLs treated with Prist for 24 h. (F) Western blot analysis of p-STAT3, total STAT3, and GAPDH showing inhibition of constitutive STAT3 activation by Prist.

To determine whether Prist-mediated growth inhibition is apoptosis-driven, we assessed apoptotic markers. Flow cytometric Annexin V/PI staining showed a pronounced, dose-dependent increase in apoptotic cell populations upon Prist treatment (Figure D). Immunoblotting further revealed robust activation of the apoptotic machinery, evidenced by increased levels of active caspase-3 and PARP cleavage (Figure E).

Because constitutive STAT3 activation is a key survival mechanism in EBV-transformed cells, we next examined whether Prist affects this pathway. Indeed, Prist treatment markedly reduced STAT3 phosphorylation in B-LCLs without altering total STAT3 levels (Figure F), indicating direct inhibition of a major EBV-driven oncogenic signaling cascade.

Collectively, these findings demonstrate that Prist exerts substantial antiproliferative and pro-apoptotic effects on EBV-transformed B-LCLs. By suppressing STAT3 activation and triggering caspase-mediated apoptosis, Prist effectively counteracts EBV-induced survival signaling, supporting its potential therapeutic relevance in EBV-associated B-cell malignancies.

Discussion

MM is a clonal plasma cell malignancy characterized by bone marrow accumulation of abnormal plasma cells that exhibit profound resistance to apoptosis. , This apoptotic resistance is driven largely by dysregulated intracellular signaling pathways, most notably the JAK/STAT3 axis, which promotes cell survival, proliferation, and drug resistance. Given the limitations of current therapies, including relapse and chemoresistance, there is a continuing need for novel agents capable of modulating these survival pathways. In this context, natural compounds, particularly those with multitargeted mechanisms, have gained increasing attention for their anticancer potential. The present study provides comprehensive evidence that Prist, a naturally occurring triterpenoid, exerts potent antimyeloma activity by inducing apoptosis, disrupting mitochondrial integrity, generating reactive oxygen species (ROS), and inhibiting STAT3 signaling.

Our findings demonstrate that Prist significantly reduces MM cell viability in a dose-dependent manner, consistent with prior work showing the anticancer potential of triterpenoids in hematologic malignancies. Morphological evidence of cytotoxicity, combined with CCK-8 and live/dead staining, confirmed substantial Prist-mediated growth inhibition. Mechanistically, Prist triggered hallmark apoptotic events, including sub-G0/G1 accumulation, increased Annexin V positivity, induction of DNA double-strand breaks as marked by p-H2AX, and activation of initiator and effector caspases (caspase-8 and caspase-3). Cleavage of PARP further supported apoptosis induction. Similar results were demonstrated by our group in myeloma cell lines treated with Prist

Both intrinsic and extrinsic pathways were activated, as evidenced by caspase-8 activation and Bid truncation, key events bridging death receptor signaling with mitochondrial damage. Prist also altered the balance of Bcl-2 family proteins by increasing Bax and reducing Bcl-2 expression, a shift known to promote mitochondrial outer membrane permeabilization (MOMP). Concordantly, JC-1 staining demonstrated a clear loss of mitochondrial membrane potential (MMP), confirming Prist’s role in destabilizing mitochondrial integrity. These findings align with prior reports that natural products induce apoptosis in MM by modulating Bcl-2 family proteins and promoting mitochondrial dysfunction, , further supporting the relevance of Prist as a mitochondrial-targeting agent.

Recent studies have shown that deregulated intracellular signaling through the JAK/STAT pathway contributes to the development of resistance to apoptosis in many human malignancies. The constitutive activation of STAT3 is a defining feature of MM pathobiology, driving transcription of antiapoptotic genes such as Bcl-xL, survivin, XIAP, and Bcl-2. − Persistent STAT3 activation promotes tumor survival, proliferation, immune evasion, and resistance to chemotherapeutics. Using network pharmacology, we identified STAT3 as the top-ranked hub gene among Prist-associated and MM-related targets, highlighting its central role in MM. Molecular docking further revealed that Prist interacts with critical STAT3 SH2-domain residues such as Lys591 and Ser636sites previously established as essential for STAT3 activation in solid tumors. , Experimentally, Prist markedly inhibited STAT3 phosphorylation at Tyr705 without altering total STAT3 protein, indicating selective blockade of STAT3 activation. The reduction of downstream survival proteins, including XIAP, cIAP1, and Bcl-xL, further supports direct STAT3 pathway inhibition. Importantly, Prist also abrogated IL-6–induced STAT3 activation, a microenvironmental survival signal that contributes to MM proliferation and drug resistance. , This finding is particularly significant because IL-6–mediated STAT3 activation is a principal mechanism through which bone marrow stromal cells confer therapeutic resistance. Thus, Prist’s ability to inhibit both constitutive and cytokine-induced STAT3 activity suggests that it could overcome microenvironment-driven resistance, an essential requirement for effective MM therapy.. ROS generation is a well-recognized mechanism through which many anticancer compounds induce cell death. Our study shows that Prist robustly elevates ROS levels, and scavenging ROS with NAC effectively rescued MM cells from Prist-induced DNA damage, caspase activation, and apoptotic death. This strongly positions ROS as a pivotal upstream event in Prist’s mechanism of action. Similar observations have been reported in other triterpenoid compounds, which rely on oxidative stress to trigger mitochondrial dysfunction and apoptosis in hematologic cancers. , Therefore, the Prist–ROS–mitochondria axis represents a coherent and biologically plausible cytotoxic mechanism. Our results are consistent with our previously published articles. , A key strength of the current work is the integration of phenotypic, biochemical, and in silico analyses to support a convergent mechanism focused on ROS generation, mitochondrial dysfunction, and STAT3 pathway inhibition. Compared with previous Prist studies in solid tumors, our data expand the mechanistic relevance of Prist in MM by demonstrating inhibition of both constitutive and IL-6-stimulated STAT3 signaling, which is particularly important in the bone marrow microenvironment, where cytokine-driven survival signals limit treatment responses. Additionally, the observed synergy with BTZ places Prist within a clinically meaningful therapeutic framework rather than only as a single-agent cytotoxic compound. Meanwhile, Prist has also been reported to affect other pathways (e.g., NF-kB, MAPK/JNK, PI3K/AKT/mTOR, proteasome-related signaling) in different cancers, and these pathway interactions may also contribute to the phenotype seen in MM. Therefore, while our data strongly support a STAT3/ROS-centered mechanism, they do not rule out cooperative effects from other signaling pathways.

Resistance to BTZ, a standard-of-care proteasome inhibitor, remains a major clinical challenge in MM. Remarkably, our study demonstrates that combining subtoxic concentrations of Prist and BTZ produces strong synergistic cytotoxicity (CI < 1). This synergy was accompanied by enhanced activation of caspase-3 and caspase-8, cleavage of PARP, and suppression of STAT3 and Bid. Downregulation of STAT3 is particularly significant because STAT3 activation has been implicated in BTZ resistance. Thus, Prist may serve as a potent chemosensitizer capable of resensitizing resistant MM clones and lowering the effective dose of BTZ, thereby minimizing toxicity. Although Prist-BTZ synergy has been reported previously our study extends this observation by quantifying synergy at subtoxic low-dose conditions using CI/DRI analysis and by providing STAT3-centered mechanistic validation (p-STAT3 suppression with amplified caspase activation and PARP cleavage) that links the combination effect to pathway inhibition and apoptotic execution.

EBV-transformed B-LCLs depend on LMP1-mediated activation of JAK/STAT pathways for survival and proliferation. Our findings show that Prist suppresses viability and induces caspase-mediated apoptosis in B-LCLs while inhibiting constitutive STAT3 activation. These effects mirror those observed in MM cells and suggest that Prist’s mechanism extends beyond MM to EBV-associated B-cell malignancies. This is supported by previous studies showing that natural compounds, such as curcumin, inhibit EBV-driven lymphomas by modulating NF-kB and activating caspases. , Thus, Prist emerges as a potentially valuable therapeutic candidate for EBV-related diseases as well.

Conclusions

Our study demonstrates that Prist has strong antimyeloma effects through multiple mechanisms, including ROS production, mitochondrial disruption, caspase activation, and direct inhibition of STAT3 signaling. Prist disrupts tumor survival pathways, increases MM cells’ sensitivity to bortezomib, and reduces microenvironment-driven IL-6 signaling. Its effectiveness against EBV-transformed B-LCLs further enhances its therapeutic potential. Network pharmacology and molecular docking confirmed Prist’s plausible binding within the STAT3 SH2 domain, a key component of the JAK/STAT pathway, supporting its inhibitory role. These results strongly support further research on Prist, both alone and in combination treatments, to overcome drug resistance and improve outcomes in MM.

Model Limitations and Challenges

This study has a few limitations that should be kept in mind when interpreting the results. First, the mechanistic experiments mainly used established MM cell lines and EBV-transformed B-LCLs, which do not fully capture the complexity of patient tumors, clonal heterogeneity, or the protective bone marrow environment. Specifically, stromal-cell contact, extracellular matrix interactions, hypoxia, and cytokine gradients can significantly influence drug response and lead to resistance in MM. Additionally, EBV-transformed B-LCLs are not considered "normal controls" and their immortalization biology differs significantly from that of malignant plasma cells; therefore, findings in B-LCLs should be seen as supporting evidence that Prist can inhibit constitutive STAT3-linked survival signaling and induce apoptosis in an EBV-driven B-cell context, rather than as a direct model of MM pathogenesis or a strict comparison of MM drug sensitivity.

Second, although NAC rescue suggests an upstream role for ROS, NAC can also have broader redox and signaling effects; therefore, additional approaches (like mitochondrial ROS-specific probes, genetic modulation of antioxidant pathways, or timing-based ROS flux analyses) would help strengthen causal conclusions. Third, while network pharmacology and docking indicate STAT3 as a likely direct target, biochemical target-engagement methods (such as CETSA, pull-down/competition assays, SPR, or mutational validation of SH2-domain residues) are necessary to confirm direct binding within cells. The SH2 domain was selected based on docking predictions and established STAT3 activation biology, and direct biochemical target-engagement remains to be established. Fourth, although the combination data with BTZ show synergy in vitro, validating the clinical relevance of this combination will require testing in BTZ-resistant models, patient-derived MM cells, and coculture systems that better mimic the bone marrow microenvironment. Recent research also highlights that traditional 2D culture systems do not fully model MM drug resistance and may underestimate key resistance mechanisms, emphasizing the need for 3D, microfluidic, and organ-on-chip methods in future studies.

Another challenge for clinical translation involves drug-like properties and formulation issues. As with many natural triterpenoids, developing Prist as a clinical drug may be limited by issues such as poor solubility, incomplete pharmacokinetic data, and the need to determine an optimal therapeutic window in vivo. Recent reviews of Prist highlight its broad anticancer activity wherein polypharmacology can be advantageous in cancers like MM where survival depends on redundant and compensatory signaling networks; simultaneously targeting multiple stress-survival programs may reduce the likelihood of rapid escape. On the other hand, multipathway effects can raise concerns regarding off-target activity and toxicity in normal tissues, particularly if the same signaling nodes are important for healthy cell homeostasis. In addition to polypharmacology the articles also highlight the importance of improving formulation and delivery to advance translational progress. Overcoming these challenges through nanoformulation, PK/PD studies, and toxicity assessments will be crucial before proceeding to preclinical efficacy testing in relevant MM models.

Future Directions

Future research should evaluate Prist in primary MM patient samples, stromal coculture, 3D bone marrow-mimetic systems, and BTZ-resistant models to determine whether the observed STAT3 suppression and ROS-dependent apoptosis are maintained in more clinically relevant settings. Given MM heterogeneity, it will be important to expand sensitivity profiling across molecularly diverse MM models (including IL-6/STAT3-high settings, genetically distinct subtypes, and clinically relevant resistant states) to identify which MM contexts are most responsive and to support biomarker-guided patient stratification. In vivo studies should include pharmacokinetics, tissue exposure, and toxicity assessments, along with pharmacodynamic biomarkers (e.g., p-STAT3, cleaved PARP, oxidative stress markers) to establish mechanism-linked efficacy.

In addition, direct target-engagement studies should be pursued to confirm STAT3 binding in cells and to define whether STAT3 inhibition is necessary and sufficient for Prist-driven cytotoxicity (e.g., SH2-domain mutational validation, competition/pull-down assays, or CETSA-based stabilization). Mechanistically, ROS studies can be strengthened using orthogonal approaches that distinguish mitochondrial ROS from global oxidative stress and capture early temporal events preceding apoptosis.

Finally, while the EBV-B-LCL experiments provide supportive mechanistic evidence for STAT3-linked survival signaling suppression in an EBV-driven B-cell context, future work should test Prist in additional EBV-associated B-cell malignancy models and primary samples to clarify disease relevance without overextending inference from immortalized lines.

In summary, our study demonstrates that Prist is a potent antimyeloma agent that triggers ROS-dependent mitochondrial apoptosis and inhibits STAT3 signaling, including IL-6-driven activation, and synergizes with BTZ. By placing these findings within the wider mechanistic literature and recognizing current model and translational limitations, our data supports further preclinical development of Prist as a potential therapeutic and chemosensitizer in MM.

Glossary

Abbreviations

JAK/STAT

Janus kinase/signal transducer and activator of transcription pathway

MM

multiple myeloma

kind of malignancy

sometimes referred to as Kahler’s disease

Sub G0/G1

G0 refers for the Quiescent/Gap 0 phase (resting state) and G1 stands for Gap 1 (first growth phase)

p-STAT3

phosphorylated signal transducer and activator of transcription-3

IL-6

interleukin-6 proinflammatory mediator and anti-inflammatory myokine

BTZ

bortezomib

Bcl-2

B-cell lymphoma 2: gene and its corresponding protein that regulates apoptosis

Bcl-xl

extralarge B-cell lymphoma: strong antiapoptotic member of the Bcl-2 family

Mcl-1

leukemia myeloid-1: it is an essential prosurvival protein

NF-kB

nuclear factor kappa-activated B cells that functions as a transcription factor

MAPK

mitogen-activated protein kinase: family of serine/threonine-specific protein kinases

FoxO3a

forkhead box O3a

CCK-8

cell counting kit-8

pH2AX

phosphorylated histone H2AX (specifically phosphorylated at serine 139)

PARP

polymerase ADP-ribose: maintains genomic integrity and DNA damage repair

PenStrep

penicillin–streptomycin: combined antibiotic solution used in cell culture to prevent bacterial infections

BID

BH3-interacting domain death: proapoptotic member of the Bcl-2 protein family

XIAP

X-linked inhibitor of apoptosis protein: apoptosis protein (IAP) family

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

FBS

fetal bovine serum

EthD-1

5,5′-(4,7-diazadecamethylene)-bis­(3,8-diamino-6-phenylphenanthridinium) dichloride dihydrochloride

ATCC

American Type Culture Collection

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

PVDF

polyvinylidene difluoride membrane: nonreactive, durable thermoplastic fluoropolymer utilized for Western blotting

HBSS

Hank’s balanced salt solution: sterile buffer solution used in cell culture

PBS

phosphate-buffered saline: buffer solution

EBV

Epstein–Barr virus

B-LCL

B-lymphoblastoid cell lines

PBMCs

peripheral blood mononuclear

DMSO

dimethyl sulfoxide

IRB

Institutional Review Board

IC50

half-maximal inhibitory concentration of a drug

tBid

truncated Bid

MOMP

mitochondrial outer membrane permeabilization

EGFR

epidermal growth factor receptor antibody

MMP9

matrix metalloproteinase-9: protein that detects active or proforms of the protein in disease or inflammation

KDR

kinase insert domain receptor antibody or vascular endothelial growth factor receptor 2 (VEGFR2 or VEGFR-2) or Flk-1 (fetal liver kinase-1)

PRKACA

protein kinase CAMP-activated catalytic subunit alpha: key enzyme involved in regulating cell cycle, growth, and signaling

AR

androgen receptor protein

cIAP1

cellular inhibitor of apoptosis protein-1: member of the inhibitor of apoptosis (IAP) protein family

ROS

reactive oxygen species

NAC

N-acetylcysteine: powerful antioxidant and precursor to glutathione

CI

combination index

LMP1

latent membrane protein 1

Data available on request from the authors.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.5c00829.

• Western blot data and densitometric analysis of the Western blots (PDF)

#.

Z.M., K.S.P., and U.H. contributed equally. Z.M., K.S.P., and U.H.: data curation, formal analysis, methodology, validation, writing-original draft, and writing-review and editing; F.A.:data curation, formal analysis, and methodology. N.A., F.A., and S.E.: data curation, formal analysis, investigation, and methodology; M.S., K.M.-M., M.M., and S.D.:data curation, resources, and review and editing. A.A.B.: data analysis and writing-review and editing. S.U.: conceptualization, supervision, data curation, formal analysis, methodology, validation, writing-original draft, and writing-review and editing. All authors read and approved of the final manuscript.

HSREP:05–1011–230031, Qatar Foundation, Doha, Qatar

The authors declare no competing financial interest.