Abstract
Choline, an organic cation, is one of the biofactors that play an important role in the structure and the function of biological membranes, and it is essential for the synthesis of phospholipids. Choline positron emission tomography-computed tomography (PET/CT) provides useful information for the imaging diagnosis of cancers, and increased choline accumulation has been identified in a variety of tumors. However, the molecular mechanisms of choline uptake and choline transporters in pancreatic cancer have not been elucidated. Here, we examined molecular and functional analyses of choline transporters in human pancreatic-cancer cell line MIA PaCa-2 and the elucidation of the action mechanism behind the antitumor effect of novel choline-transporter-like protein 1 (CTL1) inhibitors, Amb4269951 and its derivative Amb4269675. CTL1 and CTL2 mRNAs were highly expressed in MIA PaCa-2 cells, and CTL1 and CTL2 proteins were localized in the plasma membrane and the intracellular compartments, respectively. Choline uptake was characterized by Na+-independence, a single-uptake mechanism, and inhibition by choline-uptake inhibitor HC-3, similar to the function of CTL1. These results suggest that the uptake of extracellular choline in MIA PaCa-2 cells is mediated by CTL1. Choline deficiency and HC-3 treatment inhibited cell viability and increased caspase 3/7 activity, suggesting that the inhibition of CTL1 function, which is responsible for choline transport, leads to apoptosis-induced cell death. Both Amb4269951 and Amb4269675 inhibited choline uptake and cell viability and increased caspase-3/7 activity. Ceramide, which is increased by inhibiting choline uptake, also inhibited cell survival and increased caspase-3/7 activity. Lastly, both Amb4269951 and Amb4269675 significantly inhibited tumor growth in a mouse-xenograft model without any adverse effects such as weight loss. CTL1 is a target molecule for the treatment of pancreatic cancer, and its inhibitors Amb4269951 and Amb4269675 are novel lead compounds.
Highlights
Pancreatic cancer is difficult to differentiate in its early stages and is known to have poor prognosis
We examined the effects of choline deficiency and the HC-3-induced inhibition of choline transporter functions on cell viability and caspase-3/7 activity in MIA PaCa-2 cells
We examined the effects of HC-3, Amb4269951, and Amb4269675 (Figure 5) on [3H]choline up2.t5a.kIenhinibMitIoAryPEafCfeac-t2ocfeHllsC(-F3i,gAumreb64)2.6H99C51-3ainsdaApmosbi4ti2v6e9c6o7n5toronl[f3Hor]Cchhoolliinnee-Uuppttaakkee iinnhMibIiAtorPsaaCnad-2 wCaselrlesported to inhibit choline-transporter-like protein 1 (CTL1)-mediated choline uptake in cancer cells [7,8,9,10,11,12]
Summary
Pancreatic cancer is difficult to differentiate in its early stages and is known to have poor prognosis. CTL1-mediated choline transport is thought to be an essential function of phospholipid synthesis, a component of the plasma membrane Inhibition of these choline-uptake functions was reported to induce apoptotic cell death [8,9,10,11,12]. We searched for compounds that inhibit both CTL1-mediated choline uptake and cell viability in human pancreatic-cancer cells from a library of plant-derived natural organic compounds and discovered isoquinoline derivative Amb4269951 and its derivative Amb4269675 [19]. We examined the effects of HC-3, Amb4269951, and Amb4269675 (Figure 5) on [3H]choline up2.t5a.kIenhinibMitIoAryPEafCfeac-t2ocfeHllsC(-F3i,gAumreb64)2.6H99C51-3ainsdaApmosbi4ti2v6e9c6o7n5toronl[f3Hor]Cchhoolliinnee-Uuppttaakkee iinnhMibIiAtorPsaaCnad-2 wCaselrlesported to inhibit CTL1-mediated choline uptake in cancer cells [7,8,9,10,11,12]. Since Amb4269951 was reported to enhance caspase-3/7 activity and suppress cell viability in glSioinmcea Acemllsb4[22609],9w51eweaxsamreipnoerdtetdhetsoeeenffheacntcseocfaAspmasbe4-236/799a5c1tivanitdy aAnmdbs4u2p6p9r6e7s5s icnelMl vIiAabPilaitCyai-n2 gcleilolsm. aBoctehllsA[m20b]4,2w69e5e1xaanmdinAemdbt4h2e6s9e6e7f5feicnthsiboifteAdmcbel4l2v6i9a9b5il1itaynidn aAcmobn4ce2n69tr6a7t5ioinn-dMepIAenPdaeCnta-m2acnenllesr., BaontdhthAemirbe4ff2e6c9ts51waenredeAnhmabn4c2e6d9i6n7a5tirnehatibmiteendt-ctiemllev-idaebpileintydeinntamcoanncneenrt(rFaitgiuonre-d7eAp,Ben).dBeontthmAamnbn4e2r6, 9a9n5d1 t(hFeigiruereff7eCct)sawnderAe menbh4a2n6c9e6d75in(Faigtureraet7mDe)nsti-gtnimifiec-adnetplyenindheinbtitmedacnenllevr i(aFbigiluitryea7nAd,Bsi)g.nBiofitchanAtlmybin4c2r6e9a9s5e1d (cFaisgpuarsee-73C/7) aacntidvitAyminba42c6o9n6c7e5nt(rFaitgiounr-ede7pDe)ndsiegnntifmicaannntleyr. inhibited cell viability and significantly increased caspase-3/7 activity in a concentration-dependent manner
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