Flavokawain derivative FLS induced G2/M arrest and apoptosis on breast cancer MCF-7 cell line

Figures & data

Table 1 Comparison values of IC₅₀ of FLS, FLA, and FLB in MCF-7, MDA-MB-231 and MCF-10A cells

Cell lines	FLS (µM)			FLA (µM)^Citation9	FLB (µM) (Abu et al)^Citation10
	24 hours	48 hours	72 hours	72 hours	72 hours
MCF-7	40.90±4.50	36.36±3.10	33.30±2.50	25.13±3.00	33.76±1.35
MDA-MB-231	63.64±3.80	51.52±4.20	48.48±3.40	17.49±1.50	12.31±1.22
MCF-10A	>180	>180	>180	>100	45
Selective index of MCF-10A/MCF-7	>4.40	>4.95	>5.41	3.98	1.33
Selective index of MCF-10A/MDA	>2.83	>3.49	>3.71	5.72	3.66

Notes: Adapted from Abu N, Akhtar MN, Yeap SK, et al. Flavokawain A induces apoptosis in MCF-7 and MDA-MB231 and inhibits the metastatic process in vitro. PLoS One. 2014;9:e105244.⁹ Adapted from Abu N, Akhtar M, Yeap S, et al. Flavokawain B induced cytotoxicity in two breast cancer cell lines, MCF-7 and MDA-MB231 and inhibited the metastatic potential of MDA-MB231 via the regulation of several tyrosine kinases in vitro. BMC Complem Altern M. 2016;16:86.¹⁰

Abbreviations: FLS, (E)-1-(2′-Hydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one; FLA, flavokawain A; FLB, flavokawain B.

Table 2 The accession number and sequence of the primers used in the quantitative real-time PCR assay

Accession number	Gene	Sequence
NM_001101.3	ACTB	F: 5′-AGAGCTACGAGCTGCCTGAC-3′
		R: 5′-AGCACTGTGTTGGCGTACAG-3′
NM_002046.4	GAPDH	F: 5-GGATTTGGTCGTATTGGGC-3
		R: 5-TGGAAGATGGTGATGGGATT-3
HQ387008.1	18S rRNA	F: 5-GTAACCCGTTGAACCCCATT-3
		R: 5-CCATCCAATCGGTAGTAGCG-3
NM_005030.3	PLK1	F: 5-CCTGCACCGAAACCGAGTTAT-3
		R: 5-CCGTCATATTCGACTTTGGTTGC-3
NM_001143976.1	WEE-1	F: 5-GGGAATTTGATGTGCGACAG-3
		R: 5-CTTCAAGCTCATAATCACTGGCT-3
NM_006516.2	GLUT1	F: 5-ACAACACTGGAGTCATCAATGC-3
		R: 5-CCACAGAGAAGGAGCCAATCA-3

Figure 1 The molecular structures of FLS, flavokawain A and flavokawain B.

Abbreviation: FLS: (E)-1-(2′-Hydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one.

Figure 2 Light microscopic images of MCF-7.

Notes: (A) Untreated (control) and (B) FLS treated (36 μM at 72 hours). Scanning electron microscopy images of MCF-7 (C) untreated (control) and (D) FLS treated (36 μM at 72 hours). Acridine orange/propidium iodide (AO/PI) double staining of MCF-7 (E) untreated (control) and (F) FLS treated (36 μM at 72 hours) and (G) Bar chart analysis of the percentage of viable, apoptotic and necrotic MCF-7 of untreated (control) and FLS treated (36 μM at 72 hours) via AO/PI qualitative analysis at each time point. The experiments were done in triplicate and all data are expressed as mean ± SE with (*P<0.05). Figures shown were representative of at least three independent replicates with similar parameters.
Abbreviations: FLS: (E)-1-(2′-Hydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one; SE, standard error of the mean.

Figure 3 Flow cytometry cell cycle and Annexin V analyses of control and FLS treated MCF-7 cells.

Notes: (A) Histogram analysis of cell cycle and (B) bar chart showing the percentage of cell in each phase of the cell cycle in MCF-7 for 24, 48, and 72 hours when treated with IC₅₀ dose of FLS (36 μM). (C) Histogram analysis of Annexin V/FITC and (D) bar chart showing the percentage of cell stained with FITC and PI in MCF-7 treated with IC₅₀ dose of FLS (36 μM) for 24, 48, and 72 hours. The experiments were done in triplicate and all data are expressed as mean ± SE with (*P<0.05). Sub-G₀/G₁ is sub-Gap0/Gap1 phase. IC₅₀ represents the concentration of FLS reduced 50% of MCF-7 cells viability.
Abbreviations: FLS: (E)-1-(2′-Hydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one; SE, standard error of the mean; FITC+/PI+, late apoptosis population; FITC−/PI−, healthy cell population; FITC+/PI−, early apoptosis population; S, synthesis; RFU, relative fluorescence unit.

Figure 4 qPCR analysis of apoptosis and cell cycle related genes; PLK1, WEE-1, c-Jun, GLUT in MCF-7 treated with FLS (36 μM) for 24, 48, and 72 hours.

Note: The experiment was done in triplicate and the data are expressed as mean ± SE with (*P<0.05).
Abbreviations: FLS: (E)-1-(2′-Hydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one; SE, standard error of the mean; qPCR, quantitative real time polymerase chain reaction.

Figure 5 Differential protein expression of untreated and FLS treated MCF-7 cells.

Notes: (A) Western blot analysis of cell cycle and apoptotic-related proteins; CDC2, p-CDC2, BAX, Bcl-2, and p53 in MCF-7 treated with FLS (36 μM) for 24, 48, and 72 hours. Bar chart analysis of related proteins (B) CDC2 and p-CDC2, (C) ratio of BAX/Bcl-2 and (D) p53 after normalized against beta actin (ACTB). The experiment was done in triplicate and the data are expressed as mean ± SE with (*P<0.05). White bars with black dots is CDC2 while black bars with white dots is p-CDC2.
Abbreviations: FLS: (E)-1-(2′-Hydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one; SE, standard error of the mean.

Figure 6 Detection of the activation of caspase 9 and cytochrome c in MCF-7 treated with FLS (36 μM) for 24, 48, and 72 hours.

Note: The experiment was done in triplicate and the data are expressed as mean ± SE with (*P<0.05).
Abbreviations: FLS: (E)-1-(2′-Hydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio) phenyl)prop-2-ene-1-one; SE, standard error of the mean.

AbuNAkhtarMNYeapSKFlavokawain A induces apoptosis in MCF-7 and MDA-MB231 and inhibits the metastatic process in vitroPLoS One20149e10524425286005

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AbuNAkhtarMYeapSFlavokawain B induced cytotoxicity in two breast cancer cell lines, MCF-7 and MDA-MB231 and inhibited the metastatic potential of MDA-MB231 via the regulation of several tyrosine kinases in vitroBMC Complem Altern M20161686

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