Necroptosis Induced by Delta-Tocotrienol Overcomes Docetaxel Chemoresistance in Prostate Cancer Cells

**Background:** Prostate cancer (PCa) is the second most common solid tumor in men and the fifth cause of cancer mortality. While docetaxel (DTX) is first-line therapy for castration-resistant prostate cancer (CRPC), approximately half of patients do not respond or eventually develop resistance, often due to defects in apoptotic pathways. Natural compounds that trigger alternative regulated cell death (RCD) forms, such as necroptosis, may overcome this resistance. Delta-tocotrienol (δ-TT), a vitamin E isoform, has previously been shown to induce apoptosis, ER stress, autophagy, and paraptosis in CRPC cells. This study investigates whether δ-TT can also induce necroptosis and whether this mechanism can overcome DTX resistance.

**Methods:** Two CRPC cell lines (DU145 and PC3) and a DTX-resistant DU145 subline (DU-DXR) were used. DU-DXR cells were generated by chronic exposure to increasing DTX concentrations (5–200 nM) over 6 months. Cell viability was assessed by MTT assay. Cell death was analyzed by Annexin V-FITC/PI flow cytometry. Protein expression of necroptosis markers (p-RIP1, p-MLKL, FLIP isoforms, caspase 8) and EMT/CSC markers (E-cadherin, vimentin, Snail, Slug, CD44, CD133) was evaluated by Western blotting. MLKL membrane translocation was assessed by membrane protein extraction and immunofluorescence. Necrostatin-1 (Nec, 50 μM) was used to inhibit RIP1-dependent necroptosis, and Z-VAD-FMK (50 μM) to inhibit caspases.

**Key Results:** δ-TT (20 μg/mL) significantly reduced viability in DU145 and PC3 cells. The pan-caspase inhibitor Z-VAD did not significantly reverse δ-TT cytotoxicity at this dose, despite blocking caspase 3 cleavage, suggesting a non-apoptotic death mechanism. Flow cytometry showed δ-TT increased necrotic cells (Annexin V⁻/PI⁺) to 14.78% in DU145 and 19.42% in PC3 vs. 6.56% and 5.30% in controls, respectively. δ-TT increased phosphorylation of RIP1 and MLKL in a time-dependent manner in both cell lines, decreased FLIP-L expression (at 18–24 h in DU145; 6–24 h in PC3), and did not cleave caspase 8. MLKL translocated from cytosol to plasma and internal membranes, confirmed by both membrane fractionation and immunofluorescence. Necrostatin-1 (50 μM) counteracted δ-TT-induced RIP1 phosphorylation, reduced necrotic cell populations, and significantly reversed δ-TT cytotoxicity in both cell lines. In combination studies, δ-TT (15 μg/mL) plus DTX (50 nM) for 24 h enhanced cytotoxicity compared to either agent alone, with increased cleaved caspase 3 levels. DU-DXR cells showed EMT markers (downregulated E-cadherin, upregulated vimentin, Snail, Slug) and increased CD44 expression. DTX (10–200 nM) did not reduce DU-DXR viability, confirming resistance. However, δ-TT (10–20 μg/mL) significantly reduced DU-DXR viability in a dose-dependent manner, similar to parental DU145 cells. δ-TT did not activate caspase 3 in DU-DXR cells but increased necrotic cells (14.10% Annexin V⁻/PI⁺) and late apoptosis (6.4% Annexin V⁺/PI⁺). Necrostatin-1 reduced δ-TT-induced necrosis, counteracted RIP1 and MLKL phosphorylation, and significantly reversed δ-TT cytotoxicity in DU-DXR cells.

**Clinical Implications:** This study demonstrates for the first time that δ-TT induces RIP1/MLKL-dependent necroptosis in CRPC cells, providing an alternative cell death pathway when apoptosis is compromised. δ-TT potentiates DTX efficacy in sensitive cells and effectively kills DTX-resistant cells through necroptosis activation. These findings position δ-TT as a potential adjuvant therapy for metastatic CRPC, particularly in patients who have developed DTX resistance. The ability to trigger non-apoptotic cell death offers a strategy to circumvent chemoresistance, though further in vivo and clinical studies are needed to validate therapeutic applicability.