Calcitriol exerts an anti‐tumor effect in osteosarcoma by inducing the endoplasmic reticulum stress response

Abstract

Osteosarcoma is the most common type of primary bone tumor, and novel therapeutic approaches for this disease are urgently required. To identify effective agents, we screened a panel of Food and Drug Administration (FDA)‐approved drugs in AXT cells, our newly established mouse osteosarcoma line, and identified calcitriol as a candidate compound with therapeutic efficacy for this disease. Calcitriol inhibited cell proliferation in AXT cells by blocking cell cycle progression. From a mechanistic standpoint, calcitriol induced endoplasmic reticulum (ER) stress, which was potentially responsible for downregulation of cyclin D1, activation of p38 MAPK, and intracellular production of reactive oxygen species (ROS). Knockdown of Atf4 or Ddit3 restored cell viability after calcitriol treatment, indicating that the ER stress response was indeed responsible for the anti‐proliferative effect in AXT cells. Notably, the ER stress response was induced to a lesser extent in human osteosarcoma than in AXT cells, consistent with the weaker suppressive effect on cell growth in the human cells. Thus, the magnitude of ER stress induced by calcitriol might be an index of its anti‐osteosarcoma effect. Although mice treated with calcitriol exhibited weight loss and elevated serum calcium levels, a single dose was sufficient to decrease osteosarcoma tumor size in vivo. Our findings suggest that calcitriol holds therapeutic potential for treatment of osteosarcoma, assuming that techniques to diminish its toxicity could be established. In addition, our results show that calcitriol could still be safely administered to osteosarcoma patients for its original purposes, including treatment of osteoporosis.

Vitamin D regulation of a SOD1-to-SOD2 antioxidative switch to prevent osteosarcoma transformation

“…These results suggest that 1,25D 3 does not modulate active cytochrome c oxidase via COXII and COX4I1 subunits at the transcriptional level. These results point to alternative mitochondrial regulators, including perhaps other components of the electron transport chain, in the regulation of ROS by vitamin D 25 .…”

“…Although CYP24A1 is commonly upregulated in cancers and thought to be one mode for vitamin D resistance 32 , this appears not to be the case for MG-63 cells treated with 100nM 1,25D 3 based on the MTT results ( Figure 1A ). This suggests other anti-cancer strategies upon high levels of vitamin D, including apoptosis 33 and/or induction of other stress pathways such as those involving the endoplasmic reticulum 25 .…”

“…Recent studies have shown that vitamin D can suppress the production of cellular ROS in MG-63 cells 25 , however the mode of action is unknown. First mitochondrial and nuclear transcription of cytochrome c oxidase subunit 2 (COXII) and subunit 4 isoform 1 (COX4I1) mRNA, respectively, were characterized after 1,25D 3 treatment over time as potential players in ROS regulation.…”

“…Given the known increase in ROS among MG-63 osteosarcoma cells 25 , it was hypothesized that antioxidant defense mechanisms may be involved in vitamin D-dependent suppression of ROS production. Specific SOD activities are correlated with tumorigenicty 16,29 , therefore the transcription of the SOD genes within 1,25D 3 -treated MG-63 cells was monitored over time.…”

Vitamin D Regulation of a SOD1-to-SOD2 Antioxidative Switch to Prevent Osteosarcoma Transformation

“…However, there are several studies that have looked at the role of vitamin D on ROS production and detoxification. For example, a recent paper has shown that supraphysiological levels of calcitriol exerts its anti-tumor effects in a mouse cell model of osteosarcoma via induction of the endoplasmic reticulum stress response with a concomitant increase in intracellular ROS 25 . In addition, cDNA microarray analysis using human prostate epithelial cells revealed that calcitriol (50nM) can promote SOD2 expression (2.01 fold increase, 24 hours) 26 , as well as the early expression of the antioxidant thioredoxin reductase 1 (TXNRD1; 2.8 fold increase, 6 hours) 27 .…”