Vitamin D3 (1,25(OH)2D3), is a naturally occurring hormone formed in a UVB-dependent, non-enzymatic reaction from the 7-dehydrocholesterol in the uppermost layer of the epidermis. The full biological activity of 1,25(OH)2D3 required two subsequent hydroxylations by the liver cytochrome P450 family 2 subfamily R member 1 (CYP2R1) and the kidney cytochrome P450 family 27 subfamily B member 1 (CYP27B1). 1,25(OH)2D3 modulated the expression of large sets of genes involved in calcium-phosphate homeostasis, differentiation, proliferation, and also in the prevention of various diseases, including cancer [[1], [2], [3], [4]]. The gene expression control is mediated by the nuclear vitamin D receptor (VDR), its partner retinoid X receptor alpha (RXRA), and many coactivators or corepressors [5,6]. Recent, studies suggested also alternative receptors for vitamin D3 and its derivatives, including retinoic acid-related orphan receptors (RORs) for the 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 [7]; liver X receptors (LXRs) for the derivatives of lumisterol and vitamin D3 [8]; and aryl hydrocarbon receptor (AHR) for the 20,23-dihydroxyvitamin D3 [9]. Furthermore, the existence of fast non-genomic pathways activated by vitamin D3 was shown and the role of protein disulfide isomerase (PDIA3) was postulated (see Zmijewski 2022 for a recent review) [[10], [11], [12]].

Multiple studies indicate that the risk of squamous cell carcinoma (SCC) developing is lower in people with high levels of 25-hydroxyvitamin D in the blood [13], and vitamin D may protect against SCC [14]. Vitamin D itself inhibits the proliferation of SCC via genomic manner, by suppressing the expression of lung cancer-associated transcript 1 (LUCAT1) which regulates mitogen protein kinase (MAPK) signaling pathway [15]; promotes the cisplatin sensitivity of oral SCC by inhibiting lipocalin 2-(LCN2) modulated NF-κB pathway [16]; upregulate cell cycle checkpoints inhibitors p21, p18, p27 [[17], [18], [19]]; upregulate negative regulators of phosphoinositide-3-kinase (PI3K) including phosphatase and tensin homolog (PTEN) [20]; downregulate pro‐inflammatory cytokines including interleukin 6 (IL‐6) and tumor necrosis factor-alpha (TNF‐α) [21] and upregulate cell-surface glycoproteins that interact with programmed death 1 (PD-1) on T cells to attenuate inflammation [22].

In recent years also, more attention has been focused on the role of mitochondria in both therapy and therapy resistance of SCC. For example, proto-oncogene 1 (ROS1) is localized in SCC mitochondria, regulates mitochondrial phenotype and bioenergetics, and enhances the invasiveness of SCC [23]. Treatment of SCC via cisplatin increases mitochondrial fission 1 (FIS1) protein level, and such protein involved in mitochondrial fragmentation was used as a new target for improving the sensitivity of chemotherapy [24]. Interestingly, in mitochondria from SCC circular RNA – pumilio RNA binding family member 1 (PUM1) was found, which can inhibit pyroptosis (an inflammatory form of programmed cell death) and it is a novel target for therapeutic approaches in SCC [25].

There is a strong link between vitamin D and mitochondria. First of all, in the inner mitochondrial membrane, there are heme-containing enzymes involved in the metabolism of vitamin D3 belonging to the cytochrome P450 family, including CYP27A1, CYP27B1, CYP24A1, and CYP11A1. Furthermore, steroid receptors, including VDR, besides their classic localizations: in the nucleus and cytoplasm, were also found in the mitochondria of human platelets [26], megakaryocytes [27], keratinocytes [28], and fibroblasts [29]. Interestingly, mitochondrial localization of VDR seems to be a common feature of proliferating cancer cell lines, rather than the fully differentiated counterparts [30], and such distribution seems to be ligand-independent. VDR has no obvious N-terminal mitochondrial import sequence thus, it was suggested that the receptor is not transported through translocase of the outer and inner mitochondrial membrane (TOM/TIM translocase), but rather through permeability transition pore (PTP) dependent pathway [28]. The potential role of mitochondrial localization of VDR is still under debate. Further, in silico analysis points to other 1,25(OH)2D3 genomic effects via interaction with VDR receptor presented in the displacement loop (D-loop) of the mitochondrial genome [31,32]. Interestingly, our recent study on purified mitochondria, derived from human astrocytoma cells (U-87 MG), has shown, that 1,25(OH)2D3 directly modulates the conductivity of the mitochondrial large-conductance calcium-regulated channel, providing a new interesting field to explore [33].

In this work, the effect of the active form of 1,25(OH)2D3 and the role of the VDR/RXRA receptor complex on the morphology and bioenergetics of mitochondria in squamous cell carcinoma (cell line A431) in comparison to immortalized HaCaT keratinocytes was investigated for the first time.