Page 3 - G. Cell differentiation. division. and death

Effect of berberine on the differentiation induction
of acute promyelocytic leukemia (APL) cells
1
Hyein Noh , Aram Lee , Jihyun Lim , and Jong-Seok Lim 1,2
1
1
2
1 Department of Biological Science, Research Institute of Women’s Health and Cellular
Heterogeneity Research Center, Sookmyung Women’s University, Seoul 04310, Republic of Korea
Abstract Figure 4 Berberine induced differentiation on RA-
resistant HL-60 cells
Acute promyelocytic leukemia (APL) is characterized by the (A) RA-resistant HL-60 cells were stained with anti-CD38
abnormal accumulation and rapid proliferation of atypical antibody. CD38 expression was analyzed by flow cytometry.
promyelocytes that have impaired differentiation. (B) WT HL-60 and two RA-resistant cells (R38+ and R38-)
Differentiation therapy with all-trans retinoic acid (ATRA) is were treated with 1 μM of ATRA for 24 h. CD11b expression
commonly applied to treat APL patients, but the long-term was analyzed using flow cytometry. (B) WT HL-60 and (C, D)
therapy with ATRA easily induces drug resistance. Berberine two RA-resistant HL-60 cells were treated with 10 μM
(BBR) is natural isoquinoline alkaloid that is known to have berberine and ATRA (0.5 and 1 μM) for 24-48 h. CD11b
antitumor properties by affecting various biological functions. expression was analyzed using flow cytometry.
In the present study, we demonstrate the anti-tumor effects of
BBR in the human APL cell line HL-60. BBR in combination
with ATRA significantly increased protein levels of
differentiation markers including PU.1. BBR also
significantly increased the population of CD11b-positive cells
and it showed a synergistic effect in combination with low
concentration of ATRA. BBR increased phosphorylation of
AMPK as HL-60 differentiation was in progress. When
compound C, an AMPK inhibitor, was combined with BBR,
the differentiation induced by BBR was significantly inhibited.
BBR also induced differentiation of the ATRA-resistant HL-
60 cell line. In conclusion, these findings suggest that BBR-
mediated differentiation of HL-60 is affected by AMPK
activation and BBR is expected to be a new therapeutic
option for APL patients.
Figure 2. AMPK inhibition attenuated the berberine-
Purpose mediated differentiation of HL-60 cells
(A) HL-60 cells were treated with berberine (10 μM) and
Because of impaired hematopoietic differentiation, induction ATRA (0.5 μM) for 15-60 min. The protein level of pAMPK
of differentiation of leukemic cells by differentiation therapy and GAPDH was measured by western blot. (B) HL-60 cells
has been proven effective for APL patients. All-trans retinoic were treatedwith AICAR(2.5and 5 mM) andATRA(0.25
acid (ATRA) is a commonly used differentiation-inducing and 0.5 μM) for 48 h. (A) CD11b expression was analyzed
agent to treat APL patients. However, ATRA resistance using flow cytometry. (C) HL-60 cells were treated with
commonly occurs in patients treated continuously with ATRA Compound C (0.5 and1μM) andATRA (0.5and 1μM) for
as single agent. Combination of ATRA with anti-cancer 48 h. (A) CD11b expression were analyzed using flow
chemotherapy can reduce the relapse rate while maintaining cytometry (D) HL-60 cells were treated with Compound C
the complete remission (CR) rate, resulting in improved (0.5 and 1 μM) and berberine (5 and 10 μM) for 48 h. CD11b Figure 5. CD38 expression in HL-60 cells was up-
treatment performance. Berberine has various anti-cancer expression was analyzed using flow cytometry. ** p <0.01, regulated by berberine
effects, including inhibition of proliferation and metastasis, *** p <0.001. (A) Wile type HL-60 was treated with 0.5 μM of ATRA and
induction of apoptosis and differentiation. However, studies berberine (5 and 10 μM) for 48 h. CD38 expression was
on the differentiation-inducing effects of berberine in AML analyzed using flow cytometry. (B) R38- and (C) R38+, two
are still insufficient. The main purpose of this study was to RA-resistant HL-60 cell lines, were treated with 1 μM of
identify differentiation inducing effects of berberine in APL ATRA and berberine (5 and 10 μM) for 48 h. CD38
and reveal its molecular mechanisms. expression was analyzed using flow cytometry.
Results Conclusion

1. Berberine increases expression of PU.1 and population of
Figure 3. Effect of AMPK α1 overexpression on CD11b positive cell in HL-60 cells.
differentiation of HL-60 cells
HL-60 cells were transiently transfected with the vector 2. Differentiation, which is induced by berberine, is affected
control (mock) or AMPK α1 WT plasmid DNA. (A) The by AMPK activation indirectly.
experimental schedule for transfection. HL-60 cells were
transfected with plasma DNA for 18 h and treated with 3. Berberine increases expression of CD38, which is
DMSO or ATRA (1 μM) for 24 h. (B, C) The protein levels involved in drug resistance, during differentiation.
of AMPK, pAMPK and GAPDH were analyzed by western
blot. (C, D) The protein levels of PU.1 were analyzed by
western blot. CD11b expression was analyzed using flow References
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