Marta R. Romero

The Antiviral Activities of Artemisinin and Artesunate

Traditional Chinese medicine commands a unique position among all traditional medicines because of its 5000 years of history. Our own interest in natural products from traditional Chinese medicine was triggered in the 1990s, by artemisinin-type sesquiterpene lactones from Artemisia annua L. As demonstrated in recent years, this class of compounds has activity against malaria, cancer cells, and schistosomiasis. Interestingly, the bioactivity of artemisinin and its semisynthetic derivative artesunate is even broader and includes the inhibition of certain viruses, such as human cytomegalovirus and other members of the Herpesviridae family (e.g., herpes simplex virus type 1 and Epstein-Barr virus), hepatitis B virus, hepatitis C virus, and bovine viral diarrhea virus. Analysis of the complete profile of the pharmacological activities and molecular modes of action of artemisinin and artesunate and their performance in clinical trials will further elucidate the full antimicrobial potential of these versatile pharmacological tools from nature.

OATP8/1B3-mediated Cotransport of Bile Acids and Glutathione AN EXPORT PATHWAY FOR ORGANIC ANIONS FROM HEPATOCYTES?

In cholestasis, the accumulation of organic anions in hepatocytes is reduced by transporters (multidrug resistance-associated proteins and OSTα-OSTβ) able to extrude them across the basolateral membrane. Here we investigated whether organic anion-transporting polypeptides (OATPs) may contribute to this function. Xenopus laevis oocytes expressing human carboxylesterase-1 efficiently loaded cholic acid (CA) methyl ester, which was cleaved to CA and exported. Expression of OATP8/1B3 enhanced CA efflux, which was trans-activated by taurocholate but trans-inhibited by reduced (GSH) and oxidized (GSSG) glutathione. Moreover, taurocholate and estradiol 17β-d-glucuronide, but not bicarbonate and glutamate, cis-inhibited OATP8/1B3-mediated bile acid transport, whereas glutathione cis-stimulated this process, which involved the transport of glutathione itself with a stoichiometry of 2:1 (GSH/bile acid). No cis-activation by glutathione of OATP-C/1B1 was found. Using real time quantitative reverse transcription-PCR, the absolute abundance of OATP-A/1A2, OATP-C/1B1, and OATP8/1B3 mRNA in human liver biopsies was measured. In healthy liver, expression levels of OATP-C/1B1 were ∼5-fold those of OATP8/1B3 and >100-fold those of OATP-A/1A2. This situation was not substantially modified in several cholestatic liver diseases studied here. In conclusion, although both OATP-C/1B1 and OATP8/1B3 are highly expressed, and able to transport bile acids, their mechanisms of action are different. OATP-C/1B1 may be involved in uptake processes, whereas OATP8/1B3 may mediate the extrusion of organic anions by symporting with glutathione as a normal route of exporting metabolites produced by hepatocytes or preventing their intracellular accumulation when their vectorial traffic toward the bile is impaired.

Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity.

BACKGROUND & AIMS Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4(-/-) mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts. METHODS To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts. RESULTS Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1-CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice. CONCLUSIONS ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1-CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.

Chemoprevention, chemotherapy, and chemoresistance in colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death in industrialized countries. Chemoprevention is a promising approach, but studies demonstrating their usefulness in large populations are still needed. Among several compounds with chemopreventive ability, cyclooxygenase inhibitors have received particular attention. However, these agents are not without side effects, which must be weighed against their beneficial actions. Early diagnosis is critical in the management of CRC patients, because, in early stages, surgery is curative in >90% of cases. If diagnosis occurs at stages II and III, which is often the case, neoadjuvant chemotherapy and radiotherapy before surgery are, in a few cases, recommended. Because of the high risk of recurrence in advanced cancers, chemotherapy is maintained after tumor resection. Chemotherapy is also indicated when the patient has metastases and in advanced cancer located in the rectum. In the last decade, the use of anticancer drugs in monotherapy or in combined regimens has markedly increased the survival of patients with CRC at stages III and IV. Although the rate of success is higher than in other gastrointestinal tumors, adverse effects and development of chemoresistance are important limitations to pharmacological therapy. Genetic profiling regarding mechanisms of chemoresistance are needed to carry out individualized prediction of the lack of effectiveness of pharmacological regimens. This would minimize side effects and prevent the selection of aggressive, cross-resistant clones, as well as avoiding undesirable delays in the use of the most efficient therapeutic approaches to treat these patients.

Inhibition of Na+-Taurocholate Co-transporting Polypeptide-mediated Bile Acid Transport by Cholestatic Sulfated Progesterone Metabolites

Sulfated progesterone metabolite (P4-S) levels are raised in normal pregnancy and elevated further in intrahepatic cholestasis of pregnancy (ICP), a bile acid-liver disorder of pregnancy. ICP can be complicated by preterm labor and intrauterine death. The impact of P4-S on bile acid uptake was studied using two experimental models of hepatic uptake of bile acids, namely cultured primary human hepatocytes (PHH) and Na+-taurocholate co-transporting polypeptide (NTCP)-expressing Xenopus laevis oocytes. Two P4-S compounds, allopregnanolone-sulfate (PM4-S) and epiallopregnanolone-sulfate (PM5-S), reduced [3H]taurocholate (TC) uptake in a dose-dependent manner in PHH, with both Na+-dependent and -independent bile acid uptake systems significantly inhibited. PM5-S-mediated inhibition of TC uptake could be reversed by increasing the TC concentration against a fixed PM5-S dose indicating competitive inhibition. Experiments using NTCP-expressing Xenopus oocytes confirmed that PM4-S/PM5-S are capable of competitively inhibiting NTCP-mediated uptake of [3H]TC. Total serum PM4-S + PM5-S levels were measured in non-pregnant and third trimester pregnant women using liquid chromatography-electrospray tandem mass spectrometry and were increased in pregnant women, at levels capable of inhibiting TC uptake. In conclusion, pregnancy levels of P4-S can inhibit Na+-dependent and -independent influx of taurocholate in PHH and cause competitive inhibition of NTCP-mediated uptake of taurocholate in Xenopus oocytes.

Characterization of the role of ABCG2 as a bile acid transporter in liver and placenta.

ABCG2 is involved in epithelial transport/barrier functions. Here, we have investigated its ability to transport bile acids in liver and placenta. Cholylglycylamido fluorescein (CGamF) was exported by WIF-B9/R cells, which do not express the bile salt export pump (BSEP). Sensitivity to typical inhibitors suggested that CGamF export was mainly mediated by ABCG2. In Chinese hamster ovary (CHO cells), coexpression of rat Oatp1a1 and human ABCG2 enhanced the uptake and efflux, respectively, of CGamF, cholic acid (CA), glycoCA (GCA), tauroCA, and taurolithocholic acid-3-sulfate. The ability of ABCG2 to export these bile acids was confirmed by microinjecting them together with inulin in Xenopus laevis oocytes expressing this pump. ABCG2-mediated bile acid transport was inhibited by estradiol 17β-d-glucuronide and fumitremorgin C. Placental barrier for bile acids accounted for <2-fold increase in fetal cholanemia despite >14-fold increased maternal cholanemia induced by obstructive cholestasis in pregnant rats. In rat placenta, the expression of Abcg2, which was much higher than that of Bsep, was not affected by short-term cholestasis. In pregnant rats, fumitremorgin C did not affect uptake/secretion of GCA by the liver but inhibited its fetal-maternal transfer. Compared with wild-type mice, obstructive cholestasis in pregnant Abcg2(−/−) knockout mice induced similar bile acid accumulation in maternal serum but higher accumulation in placenta, fetal serum, and liver. In conclusion, ABCG2 is able to transport bile acids. The importance of this function depends on the relative expression in the same epithelium of other bile acid exporters. Thus, ABCG2 may play a key role in bile acid transport in placenta, as BSEP does in liver.

No Correlation between the Expression of FXR and Genes Involved in Multidrug Resistance Phenotype of Primary Liver Tumors

Farnesoid X receptor (FXR) has been recently reported to enhance chemoresistance through bile acid-independent mechanisms. Thus, FXR transfection plus activation with GW4064 resulted in reduced sensitivity to cisplatin-induced toxicity. This is interesting because primary tumors of the liver, an organ where FXR is expressed, exhibit marked refractoriness to pharmacological treatment. Here we have determined whether FXR is upregulated in hepatocellular carcinoma (HCC), cholangiocarcinoma (CGC) and hepatoblastoma (HPB) and whether this is related with the expression of genes involved in mechanisms of chemoresistance. Using RT-QPCR and Taqman low density arrays we have analyzed biopsies from healthy livers or surgically removed tumors from naive patients and cell lines derived from HCC (SK-HEP-1, Alexander and Huh7), CGC (TFK1) and HPB (HepG2), before and after exposure to cisplatin at IC50 for 72 h. In liver tumors FXR expression was not enhanced but significantly decreased (healthy liver > HCC > HPB ≈ CGC). Except for CGC, this was not accompanied by changes in the proportions of FXR isoforms. Changes in 36 genes involved in drug uptake/efflux and metabolism, expression/function of molecular targets, and survival/apoptosis balance were found. Changes affecting SLC22A1, CYP2A1 and BIRC5 were shared by HCC, CGC and HPB. Similarity in gene expression profiles between cell lines and parent tumors was found. Pharmacological challenge with cisplatin induced changes that increased this resemblance. This was not dependent upon FXR expression. Thus, although FXR may play a role in inducing chemoresistance under certain circumstances, its upregulation does not seem to be involved in the multidrug resistance phenotype characteristic of HCC, CGC and HPB.

Novel artemisinin derivatives with potential usefulness against liver/colon cancer and viral hepatitis.

Antitumor and antiviral properties of the antimalaria drug artemisinin from Artemisia annua have been reported. Novel artemisinin derivatives (AD1-AD8) have been synthesized and evaluated using in vitro models of liver/colon cancer and viral hepatitis B and C. Cell viability assays after treating human cell lines from hepatoblastoma (HepG2), hepatocarcinoma (SK-HEP-1), and colon adenocarcinoma (LS174T) with AD1-AD8 for a short (6h) and long (72h) period revealed that AD5 combined low acute toxicity together with high antiproliferative effect (IC50=1-5μM). Since iron-mediated activation of peroxide bond is involved in artemisinin antimalarial activity, the effect of iron(II)-glycine sulfate (ferrosanol) and iron(III)-containing protoporphyrin IX (hemin) was investigated. Ferrosanol, but not hemin, enhanced antiproliferative activity of AD5 if the cells were preloaded with AD5, but not if both compouds were added together. Five derivatives (AD1>AD2>AD7>AD3>AD8) were able to inhibit the cytopathic effect of bovine viral diarrhoea virus (BVDV), a surrogate in vitro model of hepatitis C virus (HCV), used here to evaluate the anti-Flaviviridae activity. Moreover, AD1 and AD2 inhibited the release of BVDV-RNA to the culture medium. Co-treatment with hemin or ferrosanol resulted in enhanced anti-Flaviviridae activity of AD1. In HepG2 cells permanently infected with hepatitis B virus (HBV), AD1 and AD4, at non-toxic concentrations for the host cells were able to reduce the release of HBV-DNA to the medium. In conclusion, high pharmacological interest deserving further evaluation in animal models has been identified for novel artemisinin-related drugs potentially useful for the treatment of liver cancer and viral hepatitis B and C.

Cisplatin-induced chemoresistance in colon cancer cells involves FXR-dependent and FXR-independent up-regulation of ABC proteins.

Export pumps often limit the usefulness of anticancer drugs. Here we investigated the effect of cisplatin on the expression of ABC proteins in human colon cancer cells. Short-term incubation of Caco-2 and LS174T cells with cisplatin resulted in up-regulation of several ABC pumps, in particular MRP2 and BCRP. In partially cisplatin-resistant cells (LS174T/R) obtained by long-term exposure to cisplatin, MRP2 and BCRP up-regulation was more marked. This was further enhanced when these cells were cultured under maintained stimulation with cisplatin. The MRP2 promoter (MRP2pr) was cloned, and partially deleted constructs linked to reporter genes were generated. Transfection of LS174T and LS174T/R cells with these constructs revealed the ability of cisplatin to activate MRP2pr. The intensity of this response was dependent on the conserved MRP2pr region. Basal MRP2pr activity was higher in LS174T/R cells, in which the expression of the transcription factors c/EBPβ, HNF1α, HNF3β, and HNF4α, but not PXR, p53, c-Myc, AP1, YB-1, NRF2, and RARα was enhanced. Up-regulation was particularly high for FXR (200-fold) and SHP (50-fold). In LS174T/R cells, GW4064 induced the expression of FGF19, SHP, OSTα/β, but not MRP2 and BCRP, although the sensitivity of these cells to cisplatin was further reduced. In LS174T cells, GW4064-induced chemoresistance was seen only after being transfected with FXR+RXR, when BCRP, but not MRP2, was up-regulated. Protection of LS174T cells against cisplatin was mimicked by transfection with BCRP. In conclusion, in colon cancer cells, cisplatin treatment enhances chemoresistance through FXR-dependent and FXR-independent mechanisms involving the expression of BCRP and MRP2, respectively.

Further Characterization of the Electrogenicity and pH Sensitivity of the Human Organic Anion-Transporting Polypeptides OATP1B1 and OATP1B3

Organic anion-transporting polypeptides (OATPs) are involved in the liver uptake of many endogenous and xenobiotic compounds, such as bile acids and drugs, respectively. Using Xenopus laevis oocytes and Chinese hamster ovary (CHO) cells expressing rat Oatp1a1, human OATP1B1, or OATP1B3, the sensitivity of these transporters to extracellular/intracellular pH (pHo/pHi) and changes in plasma membrane potential (ΔΨ) was investigated. In X. laevis oocytes, nonspecific plasma membrane permeability increased only at pHo below 4.5. Above this value, both using oocytes and CHO cells, extracellular acidification affected differently the specific transport of taurocholic acid (TCA) and estradiol 17β-d-glucuronide (E217βG) by Oatp1a1 (stimulation), OATP1B1 (inhibition), and OATP1B3 (stimulation). Changes in substrate uptake in the presence of valinomycin (K+-ionophore), carbonyl cyanide 3-chlorophenylhydrazone and nigericin (protonophores), and amiloride (Na+/H+-inhibitor) and cation replacement in the medium were studied with fluorescent probes for measuring substrate uptake (cholylglycyl amidofluorescein) and changes in pHi (SNARF-4F) and ΔΨ [DilC1(5)]. The results suggest that activity of these three carriers is sodium/potassium-independent and affected differently by changes in pHo and ΔΨ: Oatp1a1 was confirmed to be an electroneutral anion exchanger, whereas the function of both OATP1B1 and OATP1B3 was markedly affected by the magnitude of ΔΨ. Moreover, electrophysiological measurements revealed the existence of a net anion influx associated to OATP1B1/OATP1B3-mediated transport of TCA, E217βG, and estrone-3-sulfate. Furthermore, a leakage of Na+ through OATP1B1 and OATP1B3, which is not coupled to substrate transport, was found. In conclusion, these results suggest that OATP1B1 and OATP1B3 are electrogenic transporters whose activity may be strongly affected under circumstances of displacement of local pH.