Bafilomycin A1（巴佛洛霉素 A1;Baf-A1）

V-ATPase inhibitor,selective and reversible，IC50 值为 4-400 nmol/mg。Bafilomycin A1，是一种大环内酯类抗 生素，一种自噬 (autophagy) 晚期阶段抑制剂。Bafilomycin A1 阻断自噬体与溶酶体的融合，并抑制培养细胞溶酶体中的酸化和蛋白质降解。Bafilomycin A1 也诱导调亡 (apoptosis)。

CAS：88899-55-2
分子式：C35H58O9
分子量：622.84
纯度：98%
存储：Store at -20°C

Background:

Bafilomycin A1 is a selective, reversible inhibitor of vacuolar H+-ATPases (V-ATPases), blocking these proton pumps in mammalian, plant, or fungal cells with an IC50 value in the 4-400 nM range.[1] It is at least 1,000-fold less potent at most other types of ATPases.[1] Bafilomycin A1 also inhibits autophagy by preventing vacuolar acidification necessary for autophagosome maturation.[2],[3]

Reference:
[1]. Bowman, E.J., Siebers, A., and Altendorf, K. Bafilomycins: A class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proceedings of the National Academy of Sciences of the United States of America 85, 7972-7976 (1988).
[2]. Yamamoto, A., Tagawa, Y., Yoshimori, T., et al. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Structure and Function 23, 33-42 (1998).
[3]. Shacka, J.J., Klocke, B.J., and Roth, K.A. Autophagy, bafilomycin and cell death: The "A-B-Cs" of plecomacrolide-induced neuroprotection. Autophagy 2(3), 228-230 (2006).

Biological activity:

In Vitro:

Bafilomycin A1 is treated to different types of membrane ATPases with the I50 of 400 nmol/mg, 4 nmol/mg and 50 nmol/mg for the vacuolar ATPases of a fungus (N. crassa), a plant (Z. mays), and an animal (bovine abrenal medulla). The I50 values refer as μmol of Bafilomycin A1 per mg of protein giving 50% inhibition of ATPase activity[1] . 

Bafilomycin A1 ((-)-Bafilomycin A1) disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and CaP60A/SERCA-dependent autophagosome-lysosome fusion[2] . 

Bafilomycin A1 at a low concentration (1 nM) effectively and specifically inhibits and kills pediatric B-cell acute lymphoblastic leukemia cells. It targets both early and late stages of the autophagy pathway, mitochondria and induces caspase-independent apoptosis. Bafilomycin A1 induces the binding of Beclin 1 to Bcl-2, which further inhibits autophagy and promotes apoptotic cell death[5] . 

The growth of the BEL-7402 hepatocellular carcinoma and HO-8910 ovarian cancer cell lines are retarded and the metastatic potential is inhibited by Bafilomycin A1. Transmission electron microscopy and assays of capsase-3 and -9 suggest that Bafilomycin A1 induces apoptosis[6] . 

Bafilomycin A1 inhibits the growth of a variety of cultured cells dose-dependently, including golden hamster embryo and NIH-3T3 fibroblasts, whether or not they are transformed, and PC12 and HeLa cells. The IC50 of Bafilomycin A1 for inhibition of cell growth ranges from 10 to 50 nM[7]

In Vivo:

Chronic treatment with low-dose Bafilomycin A1 (0.1 mg/kg) slightly inhibits the tumor volume, but the final tumor volume does not differ significantly from the control. However, chronic treatment with high dose Bafilomycin A1 (1 mg/kg) inhibits the tumor growth significantly, compared with controls, after 21 days[8]

Bafilomycin A1 (0.1 mg/kg or 1 mg/kg; i.p. daily for 3 days) extends the survival of B-cell acute lymphoblastic leukemia (BALL) xenograft mice with advanced disease[9].

Protocol:

Cell Assay:

Cells are harvested using 0.05% trypsin and suspended in culture medium containing 10% FCS, and 200 μL suspension is added to each well of a 96-well plate. Cells are cultured for 20 h for adhesion. Bafilomycin A1 is added to the wells at the final concentrations of 200, 400 and 800 nM, in triplicate. At 24, 48 and 72 h, 20 μl WST-1 is added to the cells. Following incubation at 37°C for 4 h, the plates are read to determine the optical density (OD) at 435 nm with 675 nm reference using a spectrophotometer[2]

Animal Administration:

Mice: Tumor-bearing mice are divided randomly into three experimental groups: a low-dose Bafilomycin A1 (0.1 mg/kg per day)-treated group (n=5), a high-dose Bafilomycin A1 (1 mg/kg per day)-treated group (n=5),and a control group (n=5). Tumor size is measured and tumor volume doubling time is calculated[4]

参考文献：

[1]. Yuan N, et al. Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia. Haematologica. 2015 Mar;100(3):345-56. 

[2]. Lu X, et al. Bafilomycin A1 inhibits the growth and metastatic potential of the BEL-7402 liver cancer and HO-8910 ovarian cancer cell lines and induces alterations in their microRNA expression. Exp Ther Med. 2015 Nov;10(5):1829-1834. 

[3]. Ohkuma S, et al. Inhibition of cell growth by bafilomycin A1, a selective inhibitor of vacuolar H(+)-ATPase. In Vitro Cell Dev Biol Anim. 1993 Nov;29A(11):862-6. 

[4]. Ohta T, et al. Bafilomycin A1 induces apoptosis in the human pancreatic cancer cell line Capan-1. J Pathol. 1998 Jul;185(3):324-30. 

[5]. Mauvezin C, et al. Bafilomycin A1 disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosomelysosome fusion. Autophagy. 2015;11(8):1437-1438. 

[6]. Yuan N, et al. Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia. Haematologica. 2015;100(3):345-356. 

[7]. Yoshimori T, et al. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem. 1991;266(26):17707-17712 

[8]. Bowman EJ, et al. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A. 1988;85(21):7972-7976. 

[9]. Cattani L, et al. Bafilomycin A1 and intracellular multiplication of Legionella pneumophila. Antimicrob Agents Chemother. 1997;41(1):212-214.