Estrogens are considered the major breast cancer risk factor, and the carcinogenic potential of estrogens might be attributed to DNA modification caused by derivatives formed during metabolism. $17{\beta}$-estradiol ($E_2$), the main steroidal estrogen present in women, is meta...
Estrogens are considered the major breast cancer risk factor, and the carcinogenic potential of estrogens might be attributed to DNA modification caused by derivatives formed during metabolism. $17{\beta}$-estradiol ($E_2$), the main steroidal estrogen present in women, is metabolized via two major pathways: formation of 2-hydroxyestradiol (2-OH $E_2$) and 4-hydroxyestradiol ($4-OH\;E_2$) through the action of cytochrome P450 (CYP) 1A1 and 1B1, respectively. Previous reports suggested that $2-OH\;E_2$ has putative protective effects, while $4-OH\;E_2$ is genotoxic and has potent carcinogenic activity. Thus, the ratio of $2-OH\;E_2/4-OH\;E_2$ is a critical determinant of the toxicity of $E_2$ in mammary cells. In the present study, we investigated the effects of berberine on the expression profile of the estrogen metabolizing enzymes CYP1A1 and CYP1B1 in breast cancer MCF-7 cells. Berberine treatment produced significant induction of both forms at the level of mRNA expression, but with increased doses produced 16~ to 52~fold greater induction of CYP1A1 mRNA over CYP1B1 mRNA. Furthermore, berberine dramatically increased CYP1A1 protein levels but did not influence CYP1B1 protein levels in MCF-7 cells. In conclusion, we present the first report to show that berberine may provide protection against breast cancer by altering the ratio of CYP1A1/CYP1B1, could redirect $E_2$ metabolism in a more protective pathway in breast cancer MCF-7 cells.
Estrogens are considered the major breast cancer risk factor, and the carcinogenic potential of estrogens might be attributed to DNA modification caused by derivatives formed during metabolism. $17{\beta}$-estradiol ($E_2$), the main steroidal estrogen present in women, is metabolized via two major pathways: formation of 2-hydroxyestradiol (2-OH $E_2$) and 4-hydroxyestradiol ($4-OH\;E_2$) through the action of cytochrome P450 (CYP) 1A1 and 1B1, respectively. Previous reports suggested that $2-OH\;E_2$ has putative protective effects, while $4-OH\;E_2$ is genotoxic and has potent carcinogenic activity. Thus, the ratio of $2-OH\;E_2/4-OH\;E_2$ is a critical determinant of the toxicity of $E_2$ in mammary cells. In the present study, we investigated the effects of berberine on the expression profile of the estrogen metabolizing enzymes CYP1A1 and CYP1B1 in breast cancer MCF-7 cells. Berberine treatment produced significant induction of both forms at the level of mRNA expression, but with increased doses produced 16~ to 52~fold greater induction of CYP1A1 mRNA over CYP1B1 mRNA. Furthermore, berberine dramatically increased CYP1A1 protein levels but did not influence CYP1B1 protein levels in MCF-7 cells. In conclusion, we present the first report to show that berberine may provide protection against breast cancer by altering the ratio of CYP1A1/CYP1B1, could redirect $E_2$ metabolism in a more protective pathway in breast cancer MCF-7 cells.
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제안 방법
qPCR amplification was carried out using SYBR-green detection of PCR products in real time with an ABI-7500 Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR program was initiated by 30s at 95℃ before 40 thermal cycles, each of 5s at 95℃, 30s at 55℃, and 34s at 72℃. Data were analyzed according to the 2-ΔΔCt method (Livak et al.
The following primers were used for forward and reverse: CYP1A1 (5’ TCC TGG AGA CCT TCC GAC AC 3’ and 5’ GGG TTG ACC CAT AGC TTC TG 3’), CYP1B1 (5’ CTA GGC AAA GGT CCC AGT TC 3’ and 5’ CAG CAC CGA CAG GAG TAG CA 3’), β-actin (5’ TTG CCG ACA GGA TGC AGA AGG A 3’ and 5’ AGG TGG ACA GCG AGG CCA GGA T 3’).
대상 데이터
Human breast cancer cells MCF-7 were purchased from the American Type Culture Collection (ATCC; Manassas, VA). The cells were grown in RPMI 1640 medium (Hyclone, South Logan, UT) with 10% fetal calf serum (FBS; HyClone), 100U/ml penicillin and 100μg/ml streptomycin.
데이터처리
The data were expressed as mean ± standard deviation. The results were analyzed using the one-way analysis of variance (ANOVA) test. The statistical significance between the experimental groups and their respective controls was assessed by Tukey’s post hoc test by using GraphPad Prism 4 software package (San Diego, CA, USA).
이론/모형
Data were analyzed according to the 2-ΔΔCt method (Livak et al., 2001).
Cell lysate was centrifuged at 14,000 rpm for 15 min and the supernatant was recovered. The protein concentration of the lysates was determined by BCA method. Twenty micrograms total protein was resolved in 10% SDS-polyacrylamide gels and transferred to PVDF membranes (Immobilon P, Millipore, Bedford, MA) under standard conditions.
The statistical significance between the experimental groups and their respective controls was assessed by Tukey’s post hoc test by using GraphPad Prism 4 software package (San Diego, CA, USA).
성능/효과
The results of the present study showed also that berberine strongly induced CYP1B1 mRNA expression; however, the protein levels were unchanged in all cases. The lack of correlation between the CYP1B1 transcripts and protein levels was reported also in the previous studies (Sissung et al.
후속연구
Further studies are required to analyze the ratio of 2-OH E2/4-OH E2 following exposure to berberine by using high performance liquid chromatography (HPLC) analysis in vivo and in vitro, and the mechanisms of regulation of these genes by berberine also need to be further elucidated.
참고문헌 (21)
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