Impact of calcitriol and an AKT inhibitor, AT7867, on survival of rat C6 glioma cells

Figures & data

Table 1. Analyzed gene expressions.

Gene/Protein	Related pathway	Function
VDR, DBP	Vitamin D3	Activation of vitamin D pathway and role as an adaptor molecule among different pathways
CYP27B1, CYP1A1, CYP24A1	Vitamin D3	Activation/ De-activation of vitamin D
PI3K, AKT1, MTOR, CREB	Akt and c-Myc	Cell cycle progression, survival and inhibition of apoptosis
PTEN	Akt and c-Myc	Inhibition of AKT-1 for cell cycle suspension and promotion of apoptosis
BAX, BAD, CYC-C, CASP3, APAF1	Mitochondrial Apoptosis	Apoptosis
BCL2	Mitochondrial Apoptosis	Regulation of apoptosis
EGFRvIII	Other (Cell Growth)	Vitamin D related other pathways of cell cycle and growth
ENDOG	Other apoptotic pathways Caspase independent pathways	DNA fragmentation during apoptosis
MMP3	Invasion	Epithelial cells apoptosis, invasiveness
GAPDH	As a housekeeping gene for normalisation

VDR, vitamin D receptor; DBP, vitamin D binding protein, (DBP); CYP27B1, cytochrome P450 25-hydroxyvitamin D3-1alpha-hydroxylase; CYP24A1, 25-hydroxyvitamin D3-24-hydroxylase; CYP1A1, cytochrome P450 1A1; PI3K, phosphatidylinositol-3-kinase; AKT1, serine-threonine protein kinase-1; MTOR, mammalian target of rapamycin; CREB, cyclic amp-response element binding protein; PTEN, phosphatase and tensin homolog; BAX, BCL2-associated X protein; BAD, BCL2 associated agonist of cell death; CYC-C, cytochrome-C; CASP3, caspase-3; APAF1, Apoptotic peptidase activating factor-1; BCL2; B-cell lymphoma 2; EGFRvIII, epidermal growth factor receptor; MMP3, matrix metalloproteinase; ENDOG, endonuclease G; GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase.

Figure 1. Inhibition of C6 glioma cell viability across different concentrations of AT7867 and time periods. GraphPad Prism 6 was used to analyze the inhibition of cell viability and statistical analysis was performed using Student’s t-test among treated and untreated cells. Boldface values of a, b, c show statistical significance at p < 0.05.

Figure 2. Inhibition of C6 glioma cell viability across different concentrations of calcitriol for 24 h. GraphPad Prism 6 was used to analyze the inhibition of cell viability, and statistical analysis was performed using Student’s t-test among treated and untreated cells. Boldface values of * show statistical significance at p < 0.05.

Figure 3. Combined effects of 100 nmol/L calcitriol (vitamin D) and the 0.1, 1, 10 and 100 dose interval of AT7867 (inhibitor) for 24 h. GraphPad Prism 6 was used to analyze the inhibition of cell viability and statistical analysis was performed using Student’s t-test among treated and untreated cells, p values were shown in Table 2. The significances among groups were analyzed by one-way ANOVA test and boldface values of a, b and c show statistical significance at p < 0.05 by Tukey’s post-hoc test, a is between calcitriol and inhibitor, b is between calcitriol and combined dose and c is between inhibitor and combined dose.

Table 2. MTT results of different individual and combined doses of Vitamin D and AT7867 at 24 h.

24 h incubation with		% via bility	p value
Calcitriol	100 nmol/L	84.29 ± 0.61	0.048
	50 nmol/L	83.59 ± 5.81	<0.001
	25 nmol/L	74.43 ± 0.63	0.014
	10 nmol/L	100.47 ± 1.82	0.852
	1 nmol/L	114.12 ± 3.21	0.033
	0.1 nmol/L	107.07 ± 7.44	0.023
AT7867	100 μmol/L	30.01 ± 0.26	0.002
	10 μmol/L	53.40 ± 3.29	0.012
	1 μmol/L	93.48 ± 6.60	0.422
	0.1 μmol/L	81.51 ± 6.44	0.029
Combined (100 nM calcitriol + various doses of AT7867)	100 μmol/L	32.28 ± 2.79	<0.001
	10 μmol/L	74.04 ± 2.13	0.007
	1 μmol/L	99.55 ± 3.46	0.781
	0.1 μmol/L	78.41 ± 5.47	0.022

Data, absorbance readings which were normalized to untreated controls wells, are expressed as mean ± SD. Statistical analysis was performed using Student’s t-test among treated and untreated cells and boldface values of p show statistical significance.

Figure 4. Relative expression levels expressed as Log₂2^(−ΔΔCt) of related genes across 100 nmol/L calcitriol (vitamin D), 10 µmol/L AT7867 (inhibitor), and for both calcitriol and AT7867 for 24 h. Positive log2 fold change (FC) of expressions show the upregulated genes, and negative of those show downregulation of genes.

Table 3. Relative fold changes and p values of gene expressions.

Genes	Relative fold changes, 2^(−ΔΔCт)
	100 nmol/L Calcitriol	p value	10 µM AT7867	p value	Combined doses	p value
VDR	2.43	0.215	0.57	0.037	20.63	0.007
DBP	0.01	<0.001	1.08	0.656	0.30	0.003
CYP1A1	0.01	0.007	2.08	0.057	0.17	0.004
CYP27B1	0.01	<0.001	1.28	0.004	0.67	0.026
CYP24A1	2.45	<0.001	8.00	0.180	128.44	<0.001
PI3K	0.82	0.079	0.61	0.016	0.62	0.027
AKT1	0.01	0.001	0.87	0.543	0.79	0.225
MTOR	0.01	0.003	0.49	0.002	0.34	0.004
CREB	0.01	0.001	0.45	<0.001	0.52	0.005
PTEN	0.01	<0.001	0.64	<0.001	0.54	<0.001
BAX	1.65	0.582	0.64	0.013	0.52	0.005
BAD	0.01	0.002	0.28	0.057	0.05	0.001
CYC-C	0.01	<0.001	0.50	<0.001	0.64	0.002
CASP3	0.01	<0.001	1.31	0.736	0.60	0.011
APAF1	0.01	0.006	0.60	0.017	0.48	0.001
BCL2	0.01	<0.001	0.67	<0.001	0.39	0.005
EGFRvIII	0.01	0.003	1.59	0.275	0.83	0.678
MMP3	0.01	0.001	0.79	0.324	2.54	0.040
ENDOG	0.94	0.958	0.70	0.434	0.69	0.571

Relative expressional changes (FC) were expressed as 2^(−ΔΔCt) during 24 h. FC > 1 shows upregulation, and FC < 1 shows downregulation of genes. Student’s t test was used to determine the significance of relative fold change. A p value of less than 0.05 (p < 0.05) was considered as significant. Boldface p values represent significant differences.

Data availability

All data that support the findings reported in this paper are available from the corresponding author upon reasonable request.