Vitamin D’s Active Form Triggers Cell Death and Rewires Purinergic Signaling in Melanoma Cells

Calcitriol, the hormonally active form of vitamin D, has long been suspected of harboring anticancer properties, but the precise molecular circuitry it exploits in skin cancer has remained murky. Now, a team of Brazilian researchers has mapped, in remarkable cellular detail, how this vitamin D metabolite attacks one of the most aggressive forms of skin cancer: cutaneous melanoma. Writing in the journal Medical Oncology, Gilnei Bruno da Silva and colleagues report that calcitriol kills a leading laboratory model of melanoma through mitochondrial collapse and apoptosis, while simultaneously dismantling a purinergic signaling network that tumors rely upon to shield themselves from immune attack. The findings position a molecule best known for calcium regulation as a plausible adjuvant in the fight against the deadliest skin malignancy.

Cutaneous melanoma is the most lethal and aggressive cancer of the skin, arising from malignant transformation of melanocytes and marked by rapid proliferation, high metastatic potential, and notorious resistance to conventional therapies. The latest epidemiological estimates from the International Agency for Research on Cancer counted 331,722 new cases and 58,667 deaths worldwide in 2022, and even as targeted therapies and immunotherapies have transformed the landscape, low response rates and acquired resistance continue to frustrate effective management. That unmet need is precisely why the study’s authors turned their attention to two signaling systems increasingly recognized as vulnerabilities in tumors: purinergic signaling, the extracellular communication network built on adenosine triphosphate and its breakdown products, and the inflammatory axis involving interleukin-6 and the NLRP3 inflammasome.

Purinergic signaling, first proposed in 1972, operates through P1 and P2 receptors that respond to a ladder of molecules: ATP, adenosine diphosphate, adenosine monophosphate, and adenosine. Depending on which molecules dominate the extracellular space, the same pathway can drive cell proliferation or push a cell toward apoptosis. In the tumor microenvironment, the balance tilts dangerously. Extracellular adenosine, generated in abundance, acts as a potent immunosuppressant, blunting the anti-tumor immune response. The levels of these signaling molecules are controlled largely by ectonucleotidases, membrane-bound enzymes known as CD39 and CD73, along with adenosine deaminase, which breaks adenosine down into inosine. CD39 converts ATP into ADP and AMP; CD73 then hydrolyzes AMP into adenosine. Overexpression of CD73 has been documented in pancreatic adenocarcinoma, thyroid cancer, and melanoma itself, and both CD39 and CD73 have been flagged as next-generation checkpoint targets for cancer therapy.

Against that backdrop, calcitriol offered an intriguing candidate. Previous work had shown that 1-alpha,25-dihydroxyvitamin D3 induces apoptosis in melanoma cells, enhances the anticancer effects of classical chemotherapy, inhibits proliferation and migration in breast cancer models, and can modulate IL-6 expression. Yet its mechanism of action on the purinergic system in melanoma had never been systematically explored. The research team hypothesized that calcitriol exerts a potent antineoplastic effect on cutaneous melanoma by modulating the activity and expression of ectonucleotidases, thereby rewiring the extracellular nucleotide landscape in favor of tumor cell death.

To test the hypothesis, the investigators cultured two human cutaneous melanoma cell lines, A375 and SK-MEL-28, treating them with calcitriol at 1, 10, and 50 nanomolar concentrations for 24 hours. The A375 line, which displays epithelial morphology and an invasive, metastatic profile, and SK-MEL-28, a polygonal melanoma line, were subjected to a battery of assays: MTT and fluorescence microscopy viability tests, measurements of mitochondrial transmembrane potential using the TMRE dye, detection of apoptotic bodies by acridine orange staining, wound-healing migration assays, and enzymatic quantification of ATP, ADP, and AMP hydrolysis alongside adenosine deaminase activity. Gene expression of CD39, CD73, IL-6, and NLRP3 was assessed by RT-qPCR, normalized to the housekeeping gene GAPDH and analyzed with the comparative delta-delta-CT method, with one-way ANOVA and Dunnett’s post hoc test applied throughout.

The results were striking and, in one respect, unexpectedly selective. In A375 cells, calcitriol significantly reduced viability at every concentration tested, with the MTT assay yielding P values of 0.0004 at 1 nM and below 0.0001 at 10 and 50 nM. Fluorescence microscopy independently corroborated the viability loss across all doses. Crucially, the treatment also collapsed the mitochondrial transmembrane potential of A375 cells at all concentrations, with statistical significance reaching P < 0.0001, a disruption consistent with the initiation of intrinsic apoptosis. Matching that mitochondrial signature, the treated cells showed marked nuclear fragmentation: apoptotic bodies rose significantly at all three doses, while cell counts fell in a dose-dependent manner. Wound-healing assays added another dimension, revealing that calcitriol inhibited A375 migration and wound closure at every concentration tested within just 24 hours, with P < 0.0001, a finding with obvious implications for blocking metastasis.

The SK-MEL-28 line told a different story. Calcitriol produced no significant reduction in its viability and left its mitochondrial potential untouched; at 1 nM, viability even increased slightly. The researchers point to a well-documented explanation: SK-MEL-28 cells express functionally lower levels of the vitamin D receptor than several other melanoma lines, rendering them resistant to calcitriol’s antiproliferative effects at nanomolar concentrations, with prior studies noting sensitivity only at much higher doses. The contrast between the two cell lines underscores that calcitriol’s anticancer activity depends on functional vitamin D receptor signaling, a caveat that will shape which patients might ultimately benefit from a calcitriol-based adjuvant strategy.

The deepest technical insight, however, came from the purinergic analysis. Calcitriol left ATP hydrolysis unchanged but significantly decreased ADP hydrolysis at 1 and 10 nM, with a modest increase at 50 nM, and reduced AMP hydrolysis at 10 and 50 nM. Because CD39 and CD73 jointly convert ATP down the cascade to adenosine, dampened AMP breakdown means less substrate for adenosine production. Consistent with that logic, adenosine deaminase activity, which depends on adenosine availability, dropped sharply at 10 and 50 nM. At the gene level, calcitriol downregulated CD39 expression at 10 and 50 nM and exerted a strong suppressive effect on CD73 at those same concentrations, P < 0.0001. Together, these changes describe a coherent suppression of the AMP-to-adenosine axis, the very pathway tumors exploit to generate immunosuppressive adenosine in their microenvironment. By throttling adenosine generation, calcitriol may indirectly loosen the tumor’s grip on local immunity.

The inflammatory arm of the study completed the picture. Extracellular nucleotide signaling is intimately connected to inflammation, and both IL-6 and NLRP3 have dual, sometimes controversial roles in cancer, with evidence linking them to tumor progression, chemoresistance, immune cell infiltration, and poor prognosis in melanoma. Calcitriol significantly downregulated IL-6 expression at 10 nM and 50 nM, and produced an equally robust reduction in NLRP3 gene expression, with P < 0.0001 at both doses. Given that IL-6 targeting has been proposed to abrogate melanoma growth and progression, and that NLRP3 is increasingly viewed as a therapeutic target capable of reducing chemoresistance, these transcriptional effects suggest calcitriol acts on multiple fronts at once: killing tumor cells directly, impairing their migratory capacity, starving the immunosuppressive adenosine pathway, and calming the pro-tumoral inflammatory circuitry.

The authors are careful about scope. Their analysis covered gene expression rather than protein levels, and only two cell lines were examined, which they acknowledge as a limitation given the distinct metabolic profiles of A375 and SK-MEL-28. They also stress that while the nanomolar concentrations used appear practical for in vivo applications, safety testing is required, and future animal studies should monitor calcium levels to guard against hypercalcemia, the classic toxicity of active vitamin D compounds. Encouragingly, the adjuvant concept already has clinical footprints: calcitriol has been shown to enhance the effects of cisplatin and dacarbazine in melanoma cells, to combine favorably with the VEGFR inhibitor Cediranib, and, in a prospective phase Ib study, to be well tolerated in metastatic melanoma patients receiving high-dose encapsulated calcitriol alongside temozolomide, with no significant secondary side effects reported. Combined with prior evidence that calcitriol can sensitize melanoma cells to proton beam irradiation, the new purinergic mechanism adds a mechanistic rationale to an accumulating clinical case. If subsequent in vivo work confirms that calcitriol’s ectonucleotidase modulation translates into restored anti-tumor immunity, the sunshine vitamin’s most potent form could find itself written into the melanoma treatment algorithm.

Subject of Research: Antineoplastic effects of calcitriol on cutaneous melanoma cells via purinergic signaling modulation

Article Title: Calcitriol, the active form of vitamin D, induces cell death and purinergic signaling modulation in cutaneous melanoma cells

Article References: da Silva, G. B., Narzetti, R. A., Dallagnol, P., Ozelame, B. C., Manica, D., Ramos, V. H. M., Kempka, A. P., & Bagatini, M. D. (2026). Calcitriol, the active form of vitamin D, induces cell death and purinergic signaling modulation in cutaneous melanoma cells. Medical Oncology, 43(10), Article 288. https://doi.org/10.1007/s12032-026-03412-5

Image Credits: AI Generated

DOI: 10.1007/s12032-026-03412-5

Keywords: calcitriol, vitamin D, cutaneous melanoma, purinergic signaling, ectonucleotidases, CD39, CD73, apoptosis, mitochondrial membrane potential, IL-6, NLRP3, adjuvant therapy