Shikonin compound triggers prostate cancer cell death through heme oxygenase-1 and ERK/p38 pathways

Prostate cancer remains one of the most frequently diagnosed malignancies in men worldwide, and while localised disease can often be managed successfully with surgery or radiotherapy, advanced and metastatic prostate cancer continues to pose a formidable clinical challenge. Standard options for advanced disease—chemotherapy, salvage radiotherapy, and androgen deprivation therapy—can extend survival, but resistance eventually develops and quality of life is often compromised by side effects such as urinary symptoms and sexual dysfunction. Against this backdrop, a team of researchers has turned its attention to a naturally occurring plant compound, reporting new evidence that it can drive prostate cancer cells to self-destruct through a precisely mapped molecular pathway. The findings, published in the Journal of Cellular and Molecular Medicine, add a potentially valuable candidate to the growing arsenal of plant-derived anti-cancer molecules.

The compound in question is β, β-dimethylacrylshikonin, abbreviated DMAS, a naphthoquinone pigment first isolated from the root of Onosma paniculata, a biennial herb belonging to the Boraginaceae family. Shikonin derivatives have long attracted attention in pharmacology for their anti-inflammatory and anti-cancer properties, and DMAS in particular has shown stronger cytotoxic activity than its parent compound in several cancer models. Previous studies have documented its effects in breast cancer, where it suppresses proliferation by inhibiting the NF-κB pathway and enhances sensitivity to chemotherapy; in brain cancer, where it acts synergistically with epidermal growth factor receptor inhibitors; in lung cancer, where it engages the p38 signalling pathway; and in a slowly growing form of bone cancer, accompanied by activation of ERK and AKT. What remained largely unknown was whether DMAS could similarly restrain the progression of prostate cancer, and if so, by what molecular machinery.

To answer that question, the researchers worked with two widely used human prostate cancer cell lines, DU-145 and PC-3, both of which are androgen receptor-negative or express minimal non-functional androgen receptor, making them useful models of aggressive, hormone-refractory disease. The cells were cultured under standard conditions and exposed to DMAS at concentrations ranging from 1 to 16 micromolar for 24 hours. Cell viability was assessed using a microculture tetrazolium colorimetric assay, in which metabolically active cells convert a tetrazolium salt into a coloured formazan product measurable spectrophotometrically at 563 nanometres. The results were unambiguous: DMAS diminished the viability of both cell lines in a dose-dependent manner, confirming that the compound’s tumour-suppressive behaviour extends to prostate cancer.

Having established cytotoxicity, the team next asked whether the loss of viability reflected a stall in the cell cycle, an activation of programmed cell death, or both. Flow cytometry provided the answer. By labelling cellular DNA with propidium iodide and detecting the externalisation of phosphatidylserine with fluorescein-labelled annexin V, the researchers quantified the apoptotic fraction of the cell populations. DMAS-treated cells showed a markedly increased proportion of cells that were double-positive for propidium iodide and annexin V, the classic signature of apoptosis. Cell cycle analysis revealed a substantial accumulation of cells in the sub-G1 phase—a population characterised by massive DNA fragmentation and reduced DNA content that is strongly indicative of apoptotic cell death. Together, these findings established that DMAS kills prostate cancer cells primarily by triggering apoptosis rather than by merely slowing their division.

To dissect the protein-level changes underpinning this apoptotic response, the investigators deployed a proteome profiler apoptosis array capable of simultaneously measuring 35 apoptosis-associated proteins. The patterns observed in DU-145 and PC-3 cells were strikingly consistent. Two anti-apoptotic guardians, the cellular inhibitor of apoptosis protein-1 (cIAP-1) and the X-linked inhibitor of apoptosis protein (XIAP), were downregulated following DMAS treatment, while heme oxygenase-1 (HO-1), an enzyme with well-documented roles in regulating cell death, was strongly upregulated. These alterations were particularly pronounced at the higher concentration of 8 micromolar and were confirmed by conventional immunoblotting, with signal intensities quantified by densitometry and normalised to the housekeeping protein β-actin.

The caspase evidence was equally compelling. Western blot analysis showed that DMAS treatment depleted the inactive zymogen precursors of caspase-3, caspase-8, and caspase-9 while enriching for their active, cleaved forms. In parallel, poly(ADP-ribose) polymerase-1 (PARP), a nuclear enzyme whose cleavage by caspases is a hallmark of apoptosis, was processed into its characteristic fragment. The involvement of both caspase-8 and caspase-9 suggests that DMAS engages the extrinsic death receptor pathway and the intrinsic mitochondrial pathway simultaneously, converging on caspase-3, the executioner enzyme that dismantles the cell. This coordinated activation of caspase cascades provides a coherent mechanistic account of the cell death observed by flow cytometry.

A central question remained: was HO-1 upregulation a driver of apoptosis or merely a by-product? The team addressed this with small interfering RNA (siRNA) technology, transiently knocking down HO-1 expression in both cell lines 48 hours before DMAS treatment. The outcome was decisive. Silencing HO-1 significantly diminished the DMAS-induced cleavage of caspase-3, -8, and -9, indicating that HO-1 functions upstream of and is functionally required for the caspase activation triggered by the compound. The finding is notable because HO-1’s role in cancer is famously double-edged: it is commonly overexpressed in tumours relative to adjacent healthy tissue, where it often provides a cytoprotective shield during tumour progression, yet in this prostate cancer context it acted as a promoter of cell death rather than a defender. The authors suggest that HO-1 may behave in a tissue- and stage-specific manner, a nuance that could have important implications for how HO-1-targeted therapies are designed.

The study also implicated a second regulatory layer: the mitogen-activated protein kinases (MAPKs), a family of serine/threonine kinases central to how cells translate external stimuli into decisions about proliferation, stress responses, and death. After six hours of DMAS exposure, phosphorylation assays revealed that all three canonical MAPK branches—ERK1/2, JNK, and p38—were activated in both prostate cancer cell lines. To determine which of these were functionally necessary for the apoptotic outcome, the researchers pretreated cells with selective pharmacological inhibitors before adding DMAS: U0126 to block the MEK-ERK cascade, SB203580 to inhibit p38, and JNK-IN-8 to suppress JNK. The results showed that blocking either ERK or p38 significantly reduced DMAS-induced cleavage of pro-caspase-9, -8, and -3, whereas JNK inhibition did not perturb caspase activation. This positions ERK and p38 signalling as genuine contributors to the apoptotic machinery engaged by DMAS, while JNK appears dispensable in this setting.

Taken together, the study outlines a coherent signalling circuit: DMAS treatment elevates HO-1 expression and activates ERK and p38 MAPK pathways, which together drive the cleavage and activation of caspase cascades culminating in apoptotic death of prostate cancer cells. The work is not without limitations, as the authors candidly acknowledge. All experiments were conducted in vitro, and the pharmacological behaviour of DMAS after absorption and metabolism in a living organism remains to be established; animal studies will be needed to validate the tumour-suppressive effect in vivo. Furthermore, because both cell lines lack functional androgen receptor signalling—which plays a central role in prostate cancer biology—the findings will need replication in androgen receptor-positive models to confirm generalisability, particularly given that hormone-sensitive disease represents a large share of clinical cases.

Even with those caveats, the implications are significant. Natural products have historically been among the richest sources of anti-cancer drugs, and compounds that operate through clearly defined mechanisms are far more amenable to rational development than those whose actions remain mysterious. By tracing DMAS’s effects from the whole cell down to individual caspases, kinases, and apoptotic regulators, the researchers have provided a mechanistic foundation on which future preclinical work can be built. If subsequent animal and translational studies confirm these results, DMAS or derivatives optimised for potency and pharmacokinetics could eventually find a place among complementary strategies for managing prostate cancer, a disease that continues to claim a substantial proportion of cancer-related deaths among men worldwide despite decades of therapeutic advances.