October 2005
Notes
Biol. Pharm. Bull. 28(10) 1979—1982 (2005)
1979
Reversal of P-Glycoprotein-Dependent Resistance to Vinblastine by Newly
Synthesized Bisbenzylisoquinoline Alkaloids in Mouse Leukemia P388
Cells
,c
Feng-Peng WANG,a Li WANG,b Jin-Song YANG,a Masaaki NOMURA,c,d and Ken-ichi MIYAMOTO
*
a Department of Natural Organic Chemistry, School of Pharmacy, West China University of Medical Sciences; 3–17 Ren
Ming Nan Road, Chengdu 610041, China: b The National Chengdu Center for Safety Evaluation of Traditional Chinese
c
Medicine, Sichuan University; 3–17 Ren Ming Nan Road, Chengdu 610041, China: Department of Hospital Pharmacy,
School of Medicine, Kanazawa University; 13–1 Takara-machi, Kanazawa 920–8641, Japan: and d Department of Clinical
Pharmacology, Faculty of Pharmaceutical Sciences, Hokuriku University; Ho-3 Kanagawa-machi, Kanazawa 920–1165,
Japan. Received May 23, 2005; accepted June 22, 2005
We examined the ability of partially synthesized new compounds from fangchinoline and tetrandrine to re-
verse P-glycoprotein (P-gp)-dependent multidrug resistance (MDR) in vitro and in vivo. All compound enhanced
the in vitro cyctotoxic effect of vinblastin (VBL) at 0.1 mM as potent as 10 mM verapamil against the resistant cell
line P388/ADR. The combination effect tended to be strong by substitution of bulky group, resulting 5,14-dibro-
motetrandrine (compound #9) showed the strongest effect. Compound #9 increased intracellular VBL accumula-
tion in P388/ADR cells, much stronger than verapamil, as well as cytotoxic combined effect. This mechanism
seems to inhibit the function of P-gp, but not the expression of P-gp. In combination with VBL, this compound
also synergistically prolonged the life-span of P388/ADR-bearing mice. Bisbenzylisoquinoline alkaloids and their
derivatives are possible to be good candidates as modifier of MDR in cancer chemotherapy.
Key words P-glycoprotein; inhibition; vinblastine; bisbenzylisoquinoline alkaloid; 5,14-dibromotetrandrine; P388/ADR
Multidrug resistance (MDR) presents a serious problem in MATERIALS AND METHODS
cancer chemotherapy, because the tumor cells are resistant to
antitumor agents which are commonly used in clinical situa-
Materials Vinblastine (VBL), cepharanthine, verapamil
tions.1,2) The major mechanism for MDR is attributed to the and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
reduced accumulation of antitumor agents in resistant cells.3) bromide (MTT) were obtained from Sigma-Aldrich Co. (St.
It has been well known that a particular class of transmem- Louis, MO, U.S.A.). [H3]VBL sulfate (5 Ci/mmol) was pur-
brane glycoprotein (P-glycoprotein; P-gp) functions as an en- chased from Moravek Biochemicals (Brea, CA, U.S.A.). The
ergy-dependent drug-efflux pump.4) P-gp is present in both alkaloids, fangchinoline and tetrandrine, were isolated from
refractory and recurrent tumors and can be induced during the roots of Stephania tetrandra, and compounds tested were
treatment with potent and widely used antitumor drugs such synthesized as follows in West China University of Medical
as Vinca alkaloids or anthracyclines.
Sciences (Chengdu, China). Refluxing fangchilonine with
There are several chemical agents such as calcium channel 1% NaOH ethanolic solution followed by addition of corre-
blocking agents, calmodulin inhibitors and immunosuppres- sponding alkylbromide gave compounds #1 to #4. Com-
sive agents which can reverse MDR in vivo. We previously pounds #5 and #6 were obtained by reacting fangchinoline
reported that Rauwolfia alkaloids and staurospoline deriva- with propanoic anhydride and isobutylic anhydride, respec-
tives were increased intercellular accumulation of vinblastin tively, in pyridine. Compounds #7 to #9 were synthesized by
(VBL) in MDR cells.5,6) However, because these agents have halogenation of tetrandrine followed by Wiriyachitra and
strong pharmacological effects, their clinical use has been Cava.13) Compounds were used just after solubilizing in di-
limited by high concentration requirements.7) It is necessary methylsulfoxide and used the final concentration of di-
to establish proper protocols for cancer chemotherapy in methylsulfoxide in the culture medium below 0.25%.
combination with appropriate MDR-reversing agents which
Cells and Culture Mouse leukemia P388/S cells and the
have low pharmacological potencies and side effects. A bis- multidrug resistant subclone P388/ADR cells were used
benzylisoquinoline alkaloid, cepharanthine, is using for treat- (kind gifts from Dr. Inaba, Cancer Chemotherapy Center,
ment of leukopenia without severe side effects, and this drug Tokyo). Cells were maintained by weekly passage in the ab-
has been reported to increase the antitumor effect of substrate dominal cavity of CDF1 (C57BL/DBA2) mice. Cells were
drugs of P-gp in MDR tumor cells.8,9) On the other hand, suspended in RPMI1640 medium with 10% fetal calf serum,
Stephania tetrandra, containing other bisbenzylisoquinoline 100 mg/ml kanamycin and 20 mM 2-mercapthoethanol in a
alkaloids such as fangchinoline and tetrandrine, has been CO2 incubator.
used as anti-inflammatory and analgesic medicine in China,
MTT Cell Viability Assay The effects of test alkaloids
and it has been recently shown that these alkaloids enhanced on the growth inhibitory effects of VBL were evaluated by
the cytotoxicity of anticancer drugs in P-gp-dependent tumor the MTT method after 72 h culture. P388 cells (2ꢀ104) were
cells.10—12) In this study, to search for potent MDR-reversing seeded in each well of 96-well plate, after 24 h, were added a
agents from Stephania tetrandra alkaloids, we partially syn- test alkaloid at 1 h prior to varying concentration of VBL,
thesized new compounds from fangchinoline and tetrandrine and were cultured for 72 h. Subsequently, 25 ml of MTT
and examined the ability to reverse MDR in vitro and in vivo. (2 mg/ml) in phosphate-buffered saline was added to each
∗ To whom correspondence should be addressed. e-mail: miyaken@pharmacy.m.kanazawa-u.ac.jp
© 2005 Pharmaceutical Society of Japan