Assessment of synergistic effect of combining hyperthermia with irradiation and calcium carbonate nanoparticles on proliferation of human breast adenocarcinoma cell line (MCF-7 cells)

Figures & data

Table 1. Characteristics of the groups under study.

Groups	Characteristics
1	NC
2	R
3	H
4	R + H
5	50 µg/mL NPs (NP1)
6	100 µg/mL NPs (NP2)
7	150 µg/mL NPs (NP3)
8	50 µg/mL NPs + R (NP1 + R)
9	100 µg/mL NPs + R (NP2 + R)
10	150 µg/mL NPs + R (NP3 + R)
11	50 µg/mL NPs + H (NP1 + H)
12	100 µg/mL NPs + H (NP2 + H)
13	150 µg/mL NPs + H (NP3 + H)
14	50 µg/mL NPs + R+H (NP1 + R + H)
15	100 µg/mL NPs + R+H (NP2 + R + H)
16	150 µg/mL NPs + R+H (NP3 + R + H)
17	Doxorobicin 200 nM (PC1)
18	Doxorobicin 400 nM (PC2)
19	Doxorobicin 800 nM (PC3)

NC: Negative control; PC: positive control; NP: nanoparticle; R: radiotherapy; H: hyperthermia.

Figure 1. Transmission electron microscopy (TEM) image of calcium carbonate nanoparticles.

Figure 2. Standard curve of calcium carbonate nanoparticle (CC NPs) uptake into the MCF-7 cells in concentrations of 50 to 200 μg/mL.

Table 2. Mean ± standard deviations (SD) of optical densities and apoptosis in the groups under study.

Groups	Mean ± SD of variables
	MTT assay (OD)	Apoptosis (%)
NC	0.297 ± 0.013^a	17.75 ± 2.43^i
R	0.245 ± 0.014^b	27.00 ± 3.50^g
H	0.281 ± 0.012^a	21.70 ± 3.41^hi
R + H	0.243 ± 0.006^b	28.50 ± 2.62^fg
50 µg/mL NPs (NP1)	0.273 ± 0.013^a	25.75 ± 1.75^gh
100 µg/mL NPs (NP2)	0.228 ± 0.025^b	29.50 ± 2.64^efg
150 µg/mL NPs (NP3)	0.187 ± 0.025^d	34.50 ± 1.73^cd
50 µg/mL NPs + R (NP1 + R)	0.235 ± 0.017^b	28.00 ± 1.41^g
100 µg/mL NPs + R (NP2 + R)	0.195 ± 0.013^d	34.50 ± 2.38^cd
150 µg/mL NPs + R (NP3 + R)	0.156 ± 0.025^e	40.25 ± 1.50^b
50 µg/mL NPs + H (NP1 + H)	0.242 ± 0.018^b	27.50 ± 1.73^g
100 µg/mL NPs + H (NP2 + H)	0.20 ± 0.029^cd	32.75 ± 2.21^def
150 µg/mL NPs + H (NP3 + H)	0.171 ± 0.04^e	39.75 ± 1.73^b
50 µg/mL NPs + R+H (NP1 + R + H)	0.223 ± 0.025^bc	32.75 ± 3.30^de
100 µg/mL NPs + R+H (NP2 + R + H)	0.185 ± 0.008^d	38.50 ± 3.31^bc
150 µg/mL NPs + R+H (NP3 + R + H)	0.144 ± 0.017^ef	45.00 ± 1.63^a
Doxorobicin 200 nM (PC1)	0.163 ± 0.008^e	33.50 ± 1.25^de
Doxorobicin 400 nM (PC2)	0.151 ± 0.011^e	38.75 ± 2.36^bc
Doxorobicin 800 nM (PC3)	0.14 ± 0.029^f	44.50 ± 3.51^a
p values	<.05	<.05

Different superscript letters show statistically significant differences between the groups (p < .05).

Figure 3. Effects of hyperthermia (H), radiation (R), calcium carbonate nanoparticles (NPs) and doxorubicin, as positive control (PC), on the cell viability of MCF-7 cells in the studied groups. The values were normalized to those of negative control group. The Figure abbreviations are provided in Table 1. The following groups had significant differences (p < .05) as follows: NC and H with the groups labelled (A). NP1 with the groups labelled (B). R, R + H, NP1 + H and NP1 + R with the groups labelled (C). NP2 with the groups labelled (D). NP1 + R + H, NP2 + R, NP2 + H, NP3 and NP2 + R + H with the groups labelled (E). NP3 + H, PC1 and NP3 + R with the groups labelled (F).

Figure 4. The assessment of MCF-7 apoptosis by propidium iodide/acridine orange staining. The green cells with diffused chromatin are viable (A), the red cells with no condensed chromatin are necrotic (B) and the cells with condensed chromatin are apoptotic (C).

Table 3. Mean ± standard deviations (SD) of caspase-3, -8, and -9 activities in the groups under study.

Groups	Mean ± SD of variables
	Caspase-3 activity	Caspase-8 activity	Caspase-9 activity
NC	0.201 ± 0.01^m	0.294 ± 0.044^a	0.108 ± 0.026^h
R	0.251 ± 0.010^L	0.308 ± 0.028^a	0.173 ± 0.022^g
H	0.217 ± 0.013^m	0.266 ± 0.025^a	0.158 ± 0.022^gh
R + H	0.274 ± 0.028^kL	0.318 ± 0.011^a	0.221 ± 0.016^f
50 µg/ml NPs (NP1)	0.278 ± 0.023^jkl	0.328 ± 0.029^a	0.171 ± 0.018^g
100 µg/ml NPs (NP2)	0.289 ± 0.019^hijk	0.332 ± 0.036^a	0.228 ± 0.021^f
150 µg/ml NPs (NP3)	0.306 ± 0.01 4^fghjk	0.37 ± 0.043^a	0.247 ± 0.022^def
50 µg/ml NPs + R (NP1 + R)	0.323 ± 0.016^cdefg	0.351 ± 0.017^a	0.270 ± 0.018^cd
100 µg/ml NPs + R (NP2 + R)	0.314 ± 0.012^defg	0.342 ± 0.044^a	0.274 ± 0.010^c
150 µg/ml NPs + R (NP3 + R)	0.339 ± 0.021^cdefg	0.339 ± 0.031^a	0.306 ± 0.015^b
50 µg/ml NPs + H (NP1 + H)	0.28 ± 0.013^ijk	0.366 ± 0.02^a	0.230 ± 0.018^ef
100 µg/ml NPs + H (NP2 + H)	0.297 ± 0.025^ghijk	0.341 ± 0.02^a	0.235 ± 0.021^def
150 µg/ml NPs + H (NP3 + H)	0.313 ± 0.009^efg	0.331 ± 0.025^a	0.282 ± 0.015^bc
50 µg/ml NPs + R+H (NP1 + R + H)	0.342 ± 0.022^bcd	0.35 ± 0.048^a	0.372 ± 0.015^a
100 µg/ml NPs + R+H (NP2 + R + H)	0.340 ± 0.014^cd	0.358 ± 0.045^a	0.388 ± 0.02^a
150 µg/ml NPs + R+H (NP3 + R + H)	0.372 ± 0.013^b	0.336 ± 0.017^a	0.402 ± 0.036^a
Doxorobicin 200 nM (PC1)	0.357 ± 0.014^bc	0.328 ± 0.011^a	0.324 ± 0.033^g
Doxorobicin 400 nM (PC2)	0.371 ± 0.026^bc	0.359 ± 0.08^a	0.389 ± 0.012^f
Doxorobicin 800 nM (PC3)	0.446 ± 0.033^a	0.362 ± 0.04^a	0.423 ± 0.047^a
P Values	<0.05	<0.05	<0.05

Different superscript letters show statistically significant differences between the groups (P < 0.05).