../2J1(~[1merican Association for Cancer Research.
`ID
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`j
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`April 6-10) 2002 • San Francisco) California
`Volume 43 • March 2002
`
`In joint sponsorship with the Keck School of Medicine of the University of Southern California
`
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`differentiation of my11tol,d [e,u~emi~ cell lines (~~'+: Hl-60 and U937). ,S? was
`ldenllfied In our recent studies. as a potent mh1b1tor of f'TPases. Herem, we
`resent.data derncnstrattnprhat SS (250 µg/ml,-6 days) induced·B7% of NB4
`~ells to reduce nttrobtue.tetrezonum (NBT), rn comparison to the',90% induced by
`ATRA (1 µ.M, 6 days), SS-lnduced NB4 cell differentiation was confirmed by(cid:173)
`increased C011 b expression'and associated with :growth arrest at S phase and
`increased cell· death. Our results showed ·forth er it\af SS-lno1.1ced NB4 qifferen·(cid:173)
`tiation was irreversible and required continuous drug exposure for optimal induc(cid:173)
`tion. Moreover, SS (400 µ.g/ml, 6 days) induced 60% and 55% of NBT-positive
`cells in HL-60 and U937 cell lines, which were augmented in the presence of
`GM-CSF (2 ng/ml) to levels (85% and 81 %, respectively) comparable to those
`induced by ATRA. These results provide the first evidence of a differentiation
`induction activity of PTPase inhibitor SS in myeloid leukemia cell lines and
`55uggest its potential therapeutic use in myeloid leukemia. Since SS induces
`differentiation via targeting PTPases, a mechanism distinct from that of ATRA, it
`may be particularly useful in AML cases unresponsive or developed resistance to
`ATRA.
`
`#356 NAD(P}H:quinone oxidoreductase (NQOt)-depenilent and -indepen(cid:173)
`dent cytotoxicity of potent quinone cdc25 phosphatase inhibitors. Yusheng
`Han, Hongmei Shen, Brian Carr, John S, Lazo, and Su-Shu Pan. University of
`Pittsburgh Cancer Institute, Pittsburgh, PA, and University of Pittsburgh, Pitts(cid:173)
`burgh, PA.
`A vitamin K analogue, compound 5 (Cpd5, a thioethanol naphthoquinone),
`inhibits oncogenic Cdc25 phosphatases, and arrests cell cycle progression at
`both G1 and G2/M. Recently, we evaluated >10,000 compounds in the NCI
`chemical repository for in vitro inhibition against recombinant human Cdc258
`phosphatase and identified a quinone substructure in many of the active com(cid:173)
`pounds. Bioreductive enzymes in cells, however, are known to reduce various
`quinones resulting in either detoxification or activation, Therefore, we used an
`isogenic set of human colon cancer cell lines to evaluate the effect of N001 on
`the cytotoxic activity of Cpd5 and the two most potent phosphatase inhibitors
`from the repository: NSC · 95397 (a bis-thioethanol naphthoquinone) and NSC
`663284 (a quinolinedione). The two cancer cell lines used were HCT116, which
`has intermediate N001 activity, and its mitomycin C-resistant sub-line HCT116-
`R30A (R30A), which has minimum NQbl activity. Cell survival was fii.easured by
`colony formation after 7 days drug exposure. Cell cycle arrest was· evaluated by
`flow cytometry after 6 hr drug exposure, Cpd5 had an IC50 of 2.2:!:0.3 µM for
`HCT116 and 0.23:!:0,05 µ.M for A30A, i.e. a 10-fold difference. Inclusion of
`dicoumarol (10 µM), an inhibitor of NQ01, decreased the IC50 of Cpd5 for
`HCT116 to D.24:!:0.04 µM, but had no effect on A30A. In contrast, HCT116 and
`R30A cells were equally inhibited by NSC 95375 with IC50s of 1.4 :!:0.3 µM and
`1.3.:!:0.2 µM, respectively. Similarly, HCT116 and A30A cells were equally inhib(cid:173)
`ited by NSC 663284 with 1Ci0S of 2.4:!:0.3 µMand 2.6:!:0.5 µM, respi.!<:\l'(~ly. All
`lhtee compounds blocke_d tl'iir·two cell lines at the G2/M phase transllion; con(cid:173)
`sistent with cdc25 inhibition: Cpd5 at 2.5 µM arrested R30A cells at G2/M but 7 .5
`µM Cpd5 was needed to arrest HCT116 cells to a similar degree,.NSC 95375 and
`663284 arrested cell cycle progression at G2/M of HCT116 and R30A cells
`similarly, and did so in a concentration-dependent manner between 2.5 and 7.5
`µM. Our data imply that N001 in HCT116 cells protected cells from the action of
`Cpd5, probably by the reduction of Cpd5 to a less active hydroxylquinone. In
`contrast, both NSC 95397 end 663284 displayed cytotox)city·.that was indepen(cid:173)
`dent of NQ01 levels. (Support: NCI CA61862 and CA!8039}
`
`#357 Antitumor and anticarcinogenic action of Cpd 5: A new class of
`protein phosphatase lnlli.bltor;Siddha'rthti'Ka'r, Meifahg W!cing, 4!1etiggi1n'g Ren,
`Xiangbai Chen,.and. Brian i. Cwt. i;)niversity .ot Pillsbui:r}h, Pittsburgh, PA.
`B~c;kgroun!:l: Wf hij~e. ~hliml~ify ~yt\th~lj:ed a r:iew- s:la,ss. of )f!hibif(its of dual
`sp~-ciilcity pho~phatas~s (O~pl, wtjiclf play !lry lm·port~nl'roJ.a In cell 6yc.\e and
`signal transdu~tior,. ·€pd fr or 2-i2-merc~p'to'atti;1noIJ-3-mettiyl-1,4-naphthoqui(cid:173)
`none is one of the mos] potent. It Inhibits. DSPs (especially the Cdc25 :family) in
`li.$ue culture c.sills, and induces· tyrosine phosphorylation. of varlous DSP subs·
`slrates, .including Cdks and inhibits cell growth both }n vllro··Qnd In Vivo (JBC
`270:28304, 1995; Proc. MCR 39:224, 1998). Purpcse.In tliJs sIu:gy we e,vatu.ated
`(a) the antitumor and (b) ihe ann_carclnogenic acUvity of Cpd 5' for the llrst time.
`Me1hods: (a) JM1 hepatomas1 were' gro,vn · 1n 2- lnonth·otd FiscHer h1ale rah{by
`SUbcutaneouS' Injection on the back or iiitra'.portally In t11e liver. R~ts'Wera treated
`Wlih C::pd 5 t'!Y lntratuin_or, ::;ubc;utaneol.1$ (nearby~ite), h1tra0Jusd4!ar (distantsH~).
`ot lntiaperltoneal inJecllori, e_i\hiir as ):i sll)gle ~1gh acute dose or chronically as
`~~eral tow doses. (b) F)atswete'hi!ected lr\traperitoneally v~llh·a slngle dose.of the
`ti!Iclnogeh N-Nitrosodietliylamfne (DEN). lrnmunoslalned Uver·sections,for gluta0
`lhione-S-transferase,pl (GST,pl) detected pre-neoplastic tocl'atter S W~ks; Cpd
`s_ was Injected subcutaneously or intraperltoneally two weeks after DEN as a
`single high acute dose or chronically as several low doses, Re,sutts: (a) Cpd 5 had
`liQnificant Inhibitory effect onboth lntrahepatrc (14% of control, p<0.00000008)
`,!Jl(j subcutaneous (33%· of control, p<0.00008) tumor growth and also liad
`~nincant inhibitory effec_t when !nje.cted iritramustularty a,t a slte-dtstan! from the
`tumor (50% of controi.,p<0.002); There was no significant difference between the
`effects after acute or chronic injecll611s. However, foxfcity· was much lower with
`Chronic treatment. (b) The number ot enzyme altered toci was also significantly
`
`EXPERIMENTAUMOLECULAR THERAPEUTICS 3
`
`reduced when rats were treated with acute '(4_0% of control, p<0.00002) or
`chronic (50% of control, p<0.02) Cpd 5. Conclusions: .Cpd_ 5 had significant
`inhibitory effect on growth of tumors and foci.
`..
`·
`
`#358 Bosentan, a novel endothelin-A and -B receptor antagonist inhibits
`proliferation of malignant melanoma cells. Aleksandar Sekulic, Padma Suresh,
`Mark R. Pittelkow, and Svetomir N. Markovic. Mayo Foundation, Rochester, MN.
`Here we tested a feasibility of endothelin (FT) receptor blockade with a dual
`endothelin-A and -8 receptor (ETA-A and ETR-8) antagonist, Bosentan, as a
`novel !herapcut1c· approach tM malignant melanoma ETs 'are 21 aa ·pep\ldes
`priihatily piO<lLtced by findothe)ial cells arjd.iriiplicated Iha variety or physiologipal
`f.i.Jnctions, Bh:ii;lirig pl ET to gTR>A on vii~cJJlal' str;t,ic.t.ure.s P9telrtly, sllt'Yllll.at;f$
`anqioqenesis and, thus, likeiy plays an important role in growth of multiple
`cancers. Activation of ETR-8s, among other things, regulates melanocyte devel(cid:173)
`opment and function. We first examined patterns of ETA subtype expression on
`six established melanoma cell lines using flow cytometry and immunocytochem(cid:173)
`istry. Following this sections of primary and metastatic melanoma tissues were
`evaluated for ETRNETRB expression by immunohistochemistry. To test func(cid:173)
`tional effects Bosentan on melanoma cell proliferation, six melanoma cell lines
`were subjected. to .standard 3H-thymidi_rie incorporation assays in presence or
`absence of various concentrations of Bosentan. All examined melanoma tlssues
`(2 primary, 9 metastatic) express ETRE(albeit to different levels, whereas .E'tRA
`was- eX'prassed .to low ·Jilvets. In onty·J' metastafi¢' tunfora: In" fuhciional assay,s
`Bosentan',inhfb!tf/d proli/erattc,>n 'o.t al! exarnine<'l i;eu 'tin/is' V!ilh,the IQSO r:&i'.lgtng
`b!,llWeen 7 <Jl)d.4Q/,\9/ml; Ql1r·fesu!fs i;ugge~t lh(lt t)Jal!gO\!l)fl)'leh;ini:>.CY,l\'!S, 8/ipfe$,S
`iurictiorial '!;:'ms, .and theirJ~ejl\men) wiin Bas.eritan le.ids.to slgniflcarlt g'rowth
`inhibition. Concurrent inhibition of ETA-A and ETR-B in vivo by low toxicity, orally
`available inhibitor Bosentan might therefore prove useful as a novel mode of
`anti-melanoma therapy through simultaneous inhibition of cancer cell growth and
`process of angiogenesis.
`
`#359 In vivo activity of RAD001, an orally active rapamycin derivative, in
`experimental tumor models. Terence O'Reilly, Juliane Vaxelaire, Melanie Muller,
`Heinz-Herbert Fiebig, Marc Hattenberger, and Heidi A. Lane. Business Unit. On(cid:173)
`c;'ology; Novartis Pharma AG, Basel, Switzerland, and Oncotest gmbH, Freiberg,
`Gemlliny,
`.
`RA0001 is a hydroxye_ttiyl ether deriil~tjile of rapamycin th,at is pr.illy bioavail(cid:173)
`able. Rb.0001 has dem6nsir.:tted In vitro antJ0proli(9l'il.live a:ctivity igainst a panel
`of human tumor lines. For in vivo testing, tumor-bearing nude mice were admin(cid:173)
`istered RAD001 in a·.variety of doses and scheduleii. Tumors were est~l\shed by
`tra(lspiantation oHr~in:ents generated from injecilon of cells, or by tran~planta(cid:173)
`tion of fragments from stabilized_
`tumors originati_ng. from surgically removed
`H'um·an tumors. Wheii administe'reo once daify p.o., at doses-ranging from 0.5-5.0
`1ng/!(g,'oay, RAD001' was a pote·nt,inhibltor of tumor growth 'in 10 different
`xen.ograft models of-hUnian tumor~_(including panqre,1tl<;,.colon,epjde~mt)id,Jung
`and melanoma). In general, 8AD001 was well .tol.etaJed and better t<ileratfid in
`mouse xenograft models than standard cytotoxic 'ag'enis (i.e. doxbrubiciii and
`5-fluorouracil), while possessing similar antitumor activity. Only one instance of in
`viVo resistance has been observed· (MAXF 401 marru'hary l(ehograft moi::leO;
`othe/wise the activity of R,Ap(ip1 ,was generally iQhibil!011 of t1,1rnor growth [per(cid:173)
`sis,tent rllgres~ions in oneiumr:ir line, T/9,valLies of 9_ to 415 % In 8_ t1.1mor li(lesi.
`Xeriograft' mooets ·sensitive ·10 RAOcio1 treaiment rnduded tumors ex.hlb1tlng
`c6m'j5arative resistance in vitro (KB-31 and HCT116}. Persistent tumor regrllS~
`sions (41 %) were observ<'ld In a line dlspla\lfng_ sensitivity· to RAD001 in vitro
`(A549). Pharmacokinetic analyses, following a 5 mg/kg administration, indicated
`rapid uptake into plasma (Cmax 2513 ng/ml; Tmax 1 h), but the time to Cmax was
`delayed in tumors (Cmax 102 ng/g; Tmax 2 h). Elimination from the tumor (11/2,
`16 hr) was apparently slower than for plasma (11/2, 7.5 hr). RAD001 levels were
`above the IC50 of A549 cells for a 72 h period. Interestingly, tumor RAD001 levels,
`lollowing a single 5 mg/kg administration, never exceeded the in vitro antiprolif(cid:173)
`erative IC50 for either KB-31 or HCT116 cells; despite the sensitivity of these lines
`in vivo. From these observations, and given the extreme sensitivity of endothelial
`cells to RAD001, it is plausable that RAD001 may not only act on tumor cells but
`may also affec.t arigiogenesis. Taken together, these data support the application
`cii RAD001 as .an antitumor agent.
`
`#360 Discovery of anticancer agents from sponge-associated fungi. Fred(cid:173)
`erick A. Valeriote, Karen Tenney, Charles Grieshaber, Halina Pietraszkiewicz,
`Akiko Amagata. Taro Amagata, Jeff Gautschi, Joseph Media, Joseph Stayanoff,
`Richard Wiegand, and Phil Crews. Henry Ford Health System, Detroit, Ml, and
`University of California Santa Cruz, Santa Cruz, CA.
`We have evaluated 1 , 112 extracts (from 660 sponge-associated fungi) for
`assessment of potential anticancer activity. Both broth and mycelia extracts were
`assayed in most cases. Each sample was assayed in vitro against up to 8 cell
`types (murine and human) in a disk diffusion/ clonogenic assay. From these
`results, the samples were assigned into one of 4 categories: Inactive (79% of the
`extracts), Equally active across cell types (16% of the extracts), Equally active and
`potent (9 extracts or 1 %). and Solid tumor selective (42 extracts or 3.8%). The
`equally active and potent category is studied further since solid tumor selective
`compounds might exist in the extract but be concealed by one or more potent,
`cytotoxic compounds. Further, a novel, potent compound could form the basis
`for analog synthesis in an attempt to develop an active anticancer agent. One
`
`Proceedings of the American Association for Cancer Research • Volume 43 • March 2002
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