Kelussia odoratissima Mozaff. activates intrinsic pathway of apoptosis in breast cancer cells associated with S phase cell cycle arrest via involvement of p21/p27 in vitro and in vivo

Figures & data

Figure 1 ¹H NMR and chemical structures of 8-hydroxy-ar-turmerone.

Abbreviation: NMR, nuclear magnetic resonance.

Table 1 ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectral data of 8-hydroxy-ar-turmerone in CDCl₃ (δ in ppm, J in Hz)

Position	^1H NMR (δ ppm)	^13C NMR (δ ppm)
1	–	140.8
2	7.25 dd (7.2, 1.3)	129.1
3	7.13 dd (7.2, 1.3)	129.1
4	–	136.2
5	7.25 dd (7.2, 1.3)	127.6
6	7.13 dd (7.2, 1.3)	127.6
7	3.14 m	42.3
8	4.29 d (2.9)	80.5
9	–	200.2
10	6.08 d (1.1)	119.8
11	–	159.5
12	1.93 s	29.7
13	2.3 s	21.5
14	1.11 d (6.9)	13.9
15	2.21 s	28.1

Abbreviation: NMR, nuclear magnetic resonance.

Table 2 KME and 8-hydroxy-ar-turmerone were tested against MCF7, LA7 and MCF10A cell lines

Cell lines	Compounds
	8-hvdroxy-ar-turmerone (24 h)	Tamoxifen (24 h)	KME (24 h)	8-hydroxv-ar-turmerone (48 h)	Tamoxifen (48 h)	KME (48 h)	8-hvdroxv-ar-turmerone (72 h)	Tamoxifen (72 h)	KME (72 h)
MCF7 (μg/mL)	12.41±0.81	7.38±0.18	19.72±1.49	6.52±0.27	3.19±0.36	11.97±1.82	4.31±0.53	1.6±0.19	7.25±0.34
LA7 (μg/mL)	10.18±1.71	8.59±0.14	16.37±0.24	6.81±0.72	3.11±0.57	9.87±1.69	5.04±0.65	2.18±0.26	6.91±0.97
MCF10A (μg/mL)	86.91±2.16	72.38±1.72	100	72.17±3.73	65.91±2.44	100	66.15±1.76	44.83±2.37	100

Notes: The viability of the cells was determined by an MTT assay. The test agent induced cell cytotoxicity in a time-dependent manner. Dose titration curves were used to determine the IC₅₀ of the test agent toward these cell lines.

Abbreviations: IC₅₀, half maximal inhibitory concentration; KME, Kelussia odoratissima methanol extract.

Figure 2 Cytotoxicity effect of KME against different cell lines.

Abbreviations: IC₅₀, half maximal inhibitory concentration; KME, Kelussia odoratissima methanol extract.

Table 3 Reduction in tumor size in the high dose KME and tamoxifen-treated groups

Group	Treatment groups	Body weight (g)	Tumor volume (mm^3)	Reduction in tumor (%)
I	NC	292.9±15.68	0	0
II	TC	278.37±13.49	2,024±252	0
III	TT + LD	288.17±6.82	1,873±194	7.46
IV	TT + HD	274.39±14.62	521±86*	74.25*
V	TT + TAM	232.54±18.79	363±108*	82.06*

Notes: Data are shown as mean ± SEM. Values are statistically significant at

* P<0.05.

Abbreviations: HD, high dose; KME, Kelussia odoratissima methanol extract; LD, low dose; NC, normal control; SEM, standard error of the mean; TC, tumor control; TT + HD, tumor treated with HD KME; TT + LD, tumor treated with LD KME; TT + TAM, tumor treated with tamoxifen.

Figure 3 Breast cancer tumor volume in different groups.

Notes: Tumor size was significantly reduced in the HD KME- and tamoxifen-treated groups. Data are shown as mean ± SEM. Values are statistically significant at *P<0.05.
Abbreviations: HD, high dose; KME, Kelussia odoratissima methanol extract; LD, low dose; NC, normal control; SEM, standard error of the mean; TC, tumor control; TT + HD, tumor treated with HD KME; TT + LD, tumor treated with LD KME; TT + TAM, tumor treated with tamoxifen.

Figure 4 Histological examination of breast cancer tissue.

Notes: (A) Normal breast tissue. (B) Tumor control. (C) LD KME treatment. (D) HD KME treatment. (E) Tamoxifen treatment. Histological study of the breast sections shows the morphology changes of the breast tissues. Magnification, 25.2×.
Abbreviations: HD, high dose; KME, Kelussia odoratissima methanol extract; LD, low dose.

Figure 5 Apoptosis confirmation using the TUNEL assay.

Notes: (A) Tumor control. (B) LD KME treatment. (C) HD KME treatment. (D) Tamoxifen treatment. Tumor sections were subjected to TUNEL assay. The HD KME-and tamoxifen-treated groups showed remarkable numbers of apoptotic cells compared with the tumor control group. Magnification, 63×.
Abbreviations: HD, high dose; KME, Kelussia odoratissima methanol extract; LD, low dose.

Figure 6 Immunohistochemical analysis of tumor proliferation markers.

Notes: Ki-67 and PCNA are tumor markers, and brown particles with irregular shapes indicate PCNA and Ki-67 expression (A), LD and HD KME- and tamoxifen-treated groups (B). Ki-67 and PCNA quantification represents the significant reduction in tumor cells after treatment with HD KME and tamoxifen (B). Data are shown as mean ± SEM. Values are statistically significant at *P<0.05. Magnification, 100×.
Abbreviations: HD, high dose; KME, Kelussia odoratissima methanol extract; LD, low dose; SEM, standard error of the mean; TC, tumor control; TT + HD, tumor treated with HD KME; TT + LD, tumor treated with LD KME; TT + TAM, tumor treated with tamoxifen.

Figure 7 Immunohistochemical results of apoptotic markers.

Notes: Tumor control (A), LD KME treatment (B), HD KME treatment (C) and tamoxifen treatment (D). Brown particles represent Bax, Bcl-2, p53 and caspase-3 protein expression. Microscopic observation illustrates the high expression of Bax, p53 and caspase-3 and a low expression of Bcl-2 in the HD KME- and tamoxifen-treated groups. Magnification, 100×.
Abbreviations: HD, high dose; KME, Kelussia odoratissima methanol extract; LD, low dose.

Figure 8 Immunohistochemical study of cell cycle markers.

Notes: Tumor control (A), LD KME treatment (B), HD KME treatment (C) and tamoxifen treatment (D). The brown particles represent the p21 and p27 protein expression. HD KME- and tamoxifen-treated groups highlight the expression of p21 and p27 after treatment, which confirms the cell cycle arrest. Magnification, 100×.
Abbreviations: HD, high dose; KME, Kelussia odoratissima methanol extract; LD, low dose.

Figure 9 Early apoptosis validation.

Notes: MCF7 cells were treated with IC₅₀ of 8-hydroxy-ar-turmerone for 48 h. Cells were incubated for 20 min with Annexin V-FITC and the buffer. Untreated cells showed viable cells with no signs of apoptosis (A). Light green stained with Annexin V-FITC revealed that PS residues had been translocated to the membrane and externalized (phosphatidylserine) after treatment. Treated cells are shown with (B) 20× and (C) 40× magnification.
Abbreviations: FITC, fluorescein isothiocyanate; IC₅₀, half maximal inhibitory concentration.

Figure 10 Caspase-9 and caspase-7 luminescence evaluation in MCF7 cells during 8-hydroxy-ar-turmerone treatment.

Notes: Data represent a remarkable increase in caspase-9 after 12 h and caspase-7 in 72 h. This confirmed the activation of the intrinsic pathway of apoptosis after 8-hydroxy-ar-turmerone treatment. Data are shown as mean ± SD (n=3). *A significant difference (P<0.05) compared with the control.

Figure 11 Flow cytometric analysis of cell cycle distribution in MCF7 cell line.

Notes: Cells were treated with IC₅₀ of 8-hydroxy-ar-turmerone for 24, 48 and 72 h. The histogram is represented as: (A) control; (B) 24 h; (C) 48 h and (D) 74 h. Cells were cultured in RPMI 1640 (25 mL flask) media maintained at 37°C and 5% CO₂. (E) Data represents the significant elevation of the cells numbers in S phase after 48 and 72 hours of treatment. Data are shown as mean ± SD (n=3). *Significant difference (P<0.05) compared with the control.
Abbreviations: IC₅₀, half maximal inhibitory concentration; PI-A, propidium iodide A channel.