Cytotoxicity Of SN-38 Research Articles

Combination of SN-38 with gefitinib or imatinib overcomes SN-38-resistant small-cell lung cancer cells

Irinotecan is one of the effective anticancer agents for small-cell lung cancer (SCLC) and 7-ethyl-10-hydroxy-campthothecin (SN-38) is an active metabolite of irinotecan. Gefitinib and imatinib are tyrosine kinase inhibitors which have clinical activities in several malignancies and they are also potent inhibitors of breast cancer resistance protein (BCRP) transporter, which confers the resistance of topoisomerase I inhibitors including SN-38 and topotecan. The cytotoxicity of SN-38, gefitinib and imatinib for the SN-38-resistant cells (SBC-3/SN-38) from human SCLC cells, SBC-3, was evaluated using AlamarBlue assay. The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) for 96-h exposure was used to evaluate the cytotoxicity. BCRP expression was determined by Western blotting and immunofluorescence staining. Intracellular topotecan accumulation was evaluated by flow cytometry. No differences were observed in the IC50 values (mean +/- SD) of the tyrosine kinase inhibitors between the SBC-3 cells and the SBC-3/SN-38 cells: 15+/-1.6 and 12+/-2.8 microM of gefitinib, respectively; 15+/-0.51 and 14+/-3.9 microM of imatinib, respectively. The SBC-3/SN-38 was 9.5-fold more resistant to SN-38 than the parental SBC-3. The SBC-3/SN-38 restored sensitivity to SN-38 when combined with 8 microM gefitinib or 8 microM imatinib, even though the IC50 values of SN-38 combined with gefitinib or imatinib in the SBC-3 cells did not change. BCRP was equally overexpressed in the SBC-3/SN-38 with and without gefitinib or imatinib. In addition, the BCRP expression on the SBC-3/SN-38 cell membrane with and without gefitinib seemed to be equal. Gefitinib increased intracellular accumulation of topotecan in the SBC-3/SN-38 cells. Gefitinib or imatinib reversed SN-38-resistance in these SCLC cells, possibly due to intracellular accumulation of SN-38 without any change in BCRP quantity. Irinotecan with gefitinib or imatinib might be effective for SCLC refractory to irinotecan.

Antimitotic Activity and Reversal of Breast Cancer Resistance Protein‐Mediated Drug Resistance by Stilbenoids from Bletilla striata.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Estrogen-Mediated Post transcriptional Down-regulation of Breast Cancer Resistance Protein/ABCG2

Breast cancer resistance protein (BCRP)/ABCG2 mediates concurrent resistance to chemotherapeutic agents, such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan, by pumping them out of cells. We previously reported that BCRP transports sulfated estrogens. In the present study, we show that at physiologic levels, estrogens markedly decrease endogenous BCRP expression in the estrogen-responsive and estrogen receptor alpha (ERalpha)-positive human breast cancer MCF-7 cells, but not in estrogen-nonresponsive human cancer cells. 17 beta-Estradiol (E(2)) also significantly reduces exogenous BCRP expression, driven by a constitutive promoter, in BCRP-transduced estrogen-responsive and ERalpha-positive MCF-7 (MCF-7/BCRP) and T-47D cells, but not in BCRP-transduced estrogen-nonresponsive MDA-MB-231 and SKOV-3 cells. E(2) potentiates the cytotoxicity of SN-38, but not vincristine, in MCF-7/BCRP cells significantly, and increases cellular topotecan uptake in MCF-7 and MCF-7/BCRP cells. Antiestrogen tamoxifen partially reverses E(2)-mediated BCRP down-regulation in MCF-7 and MCF-7/BCRP cells and treatment of MCF-7/BCRP cells with an ERalpha small interfering RNA abolished E(2)-mediated BCRP down-regulation, suggesting that interaction of E(2) and ERalpha is necessary for BCRP down-regulation. E(2) does not affect endogenous BCRP mRNA levels in MCF-7 cells or exogenous BCRP mRNA levels in MCF-7/BCRP cells. The results from pulse-chase labeling experiments with MCF-7/BCRP cells suggest that decreased protein biosynthesis and maturation, but not alterations in protein turnover, might underlie E(2)-mediated BCRP down-regulation. These data indicate that estrogen down-regulates BCRP expression by novel posttranscriptional mechanisms. This is the first report of small molecules that can affect BCRP protein expression in cells and may therefore assist in establishing new strategies for regulating BCRP expression.

Antimitotic activity and reversal of breast cancer resistance protein-mediated drug resistance by stilbenoids from Bletilla striata

Eight stilbenoids, 1-( p-hydroxybenzyl)-4,8-dimethoxyphenanthrene-2,7-diol ( 1), 2,7-dihydroxy-1,3-bis( p-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene ( 2), 4,7-dihydroxy-1-( p-hydroxybenzyl)-2-methoxy-9,10-dihydrophenanthrene ( 3), 3,3′-dihydroxy-2′,6′-bis( p-hydroxybenzyl)-5-methoxybibenzyl ( 4), 3′,5-dihydroxy-2-( p-hydroxybenzyl)-3-methoxybibenzyl ( 5), blestriarenes B ( 6) and C ( 7), and blestrianol A ( 8) have been isolated by the guidance of inhibitory effect of tubulin polymerization from the tubers of Bletilla striata (Orchidaceae). Among them, both of bisbenzyls 4 and 5 inhibited the polymerization of tubulin at IC 50 10 μM, respectively. Furthermore bisbenzyl 4 potentiated the cytotoxicity of SN-38 in BCRP-transduced K562 (K562/BCRP) cells.

Phytoestrogens/flavonoids reverse breast cancer resistance protein/ABCG2-mediated multidrug resistance.

Breast cancer resistance protein (BCRP), also called ABCG2, confers resistance to anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan. We found previously that sulfated estrogens are physiologic substrates of BCRP. Flavonoids with weak estrogenic activities are called phytoestrogens. In this study, we show that phytoestrogens/flavonoids, such as genistein, naringenin, acacetin, and kaempferol, potentiated the cytotoxicity of SN-38 and mitoxantrone in BCRP-transduced K562 (K562/BCRP) cells. Some glycosylated flavonoids, such as naringenin-7-glucoside, also effectively inhibited BCRP. These flavonoids showed marginal effect on the drug sensitivity of K562 cells. Genistein and naringenin reversed neither P-glycoprotein-mediated vincristine resistance nor multidrug resistance-related protein 1-mediated VP-16 resistance. Genistein and naringenin increased cellular accumulation of topotecan in K562/BCRP cells. K562/BCRP cells also accumulated less [(3)H]genistein than K562 cells. [(3)H]genistein transport in the basal-to-apical direction was greater in BCRP-transduced LLC-PK1 (LLC/BCRP) cells, which express exogenous BCRP in the apical membrane, than in parental cells. Fumitremorgin C abolished the increased transport of [(3)H]genistein in LLC/BCRP cells compared with parental cells. TLC analysis revealed that genistein was transported in its native form but not in its metabolized form. These results suggest that genistein is among the natural substrates of BCRP and competitively inhibits BCRP-mediated drug efflux. The results have two important clinical implications: (a) flavonoids and glycosylated flavonoids may be useful in overcoming BCRP-mediated drug resistance in tumor cells; and (b) coadministration of flavonoids with BCRP-substrate antitumor agents may alter the pharmacokinetics and consequently increase the toxicity of specific antitumor agents in cancer patients.

Antitumor activity of new combination chemotherapy with irinotecan hydrochloride and nedaplatin against human cervical cancer cell lines.

Antitumor activity of combination chemotherapy with irinotecan hydrochloride (CPT-11) and nedaplatin was compared to that with CPT-11 and cisplatin. In vitro cytotoxicity of SN-38 (an active metabolite of CPT-11) in combination with nedaplatin or cisplatin was evaluated using three human cervical cancer cell lines (ME-180, CaSki and SiHa). IC50 values of nedaplatin against these three human cervical cancer cell lines were about 2-fold as high as those of cisplatin, indicating somewhat weak cytotoxic effects of nedaplatin. Interactions between two drugs in combination were investigated using a simultaneous-exposure schedule and analyzed by the IC50-based isobologram method. Simultaneous exposure to SN-38 with each platinum preparation showed synergistic and additive effects against ME-180 and SiHa. In vivo antitumor effects of CPT-11 in the combination with each platinum were studied using SiHa xenografts. While CPT-11, nedaplatin and cisplatin alone hardly showed any antitumor effects even at the maximum tolerated dose (MTD) levels, the combination chemotherapy with CPT-11 and nedaplatin or cisplatin resulted in significant antitumor effects even at three-quarter MTD of CPT-11 combined with two-third MTD of platinum. All treatments were tolerable for mice, indicating that the combinations did not cause significant enhancement in toxicity. In clinical application, nedaplatin causes a lower incidence of nephropathy and does not require the replacement of a large volume of fluid, which is needed for cisplatin administration, facilitating treatment at the out-patient clinic. In addition, the incidences of digestive disorder, peripheral neuropathy and auditory disorder are lower. These findings suggest that the combination chemotherapy with CPT-11 and nedaplatin for squamous cell cancer of uterine cervix is very useful in clinical practice. A dose-finding study should be conducted.

Augmented anti-metastatic efficacy of a selective matrix metalloproteinase inhibitor, MMI-166, in combination with CPT-11.

The anti-metastatic efficacy of MMI-166, which is a selective matrix metalloproteinase (MMP) inhibitor, in combination with CPT-11 was examined using two metastasis models of human gastrointestinal cancer cells. In the liver metastasis model, C-IH human colon cancer cells were injected into the spleen of athymic BALB/c nude mice. Daily oral (p.o.) dosing of MMI-166 at 200 mg/kg starting 1 day after tumor inoculation led to a significantly prolonged survival effect by inhibiting liver metastasis of C-1H tumor cells. CPT-11 (5 or 20 mg/kg) was administered intraperitoneally (i.p.) three times on day 3, day 7 and day 11 and also improved the survival of tumor-inoculated mice compared with the vehicle control. When MMI-166 was combined with CPT-11, the anti-metastatic efficacy was significantly augmented. Moreover, long tumor-free survival was noted in two of eight mice that were given the combination therapy but not either MMI-166 or CPT-11 monotherapy. In the peritoneal dissemination model, TMK-1 human gastric cancer cells were injected i.p. into nude mice. While both MMI-166, administered daily p.o. from day 1 at 200 mg/kg, and CPT-11, administered intravenously (i.v.) three times, inhibited the tumor dissemination and growth, the combination therapy of MMI-166 plus CPT-11 showed a greater inhibitory effect than each monotherapy. A hematotoxicity study demonstrated that CPT-11 alone significantly decreased the number of white blood cells (WBC) and bone marrow cells (BMC) in the mice during treatment, while the daily administration of MMI-166 alone had no such effect. More importantly, the combination therapy of MMI-166 with CPT-11 did not augment the hematotoxicity caused by CPT-11. An in vitro cytotoxicity study showed that MMI-166 itself neither has direct cytotoxicity in C-1H and TMK-1 tumor cells, nor does it augment the cytotoxicity of SN-38, an active form of CPT-11. The findings indicate that the augmented anti-metastatic efficacy in combination treatment was not simply due to the augmentation of direct cytotoxic activity, but was rather an additive or synergistic effect of anti-metastatic activities with different mechanisms. In conclusion, we demonstrated that the anti-metastatic efficacy against C-1H colon cancer and TMK-1 gastric cancer were augmented by the combination therapy of MMI-166, an orally active MMP inhibitor, with CPT-11. However, the hematotoxicity caused by CPT-11 was not augmented in the combination with MMI-166. Thus, the combination therapy of MMI-166 and CPT-11 exhibited potent anti-metastatic efficacy without increased hematotoxicity. These results point to the clinical advantage of using MMI-166 in combination with CPT-11.

Schedule-dependent cytotoxicity of SN-38 in p53 wild-type and mutant colon adenocarcinoma cell lines.

In this study the effects of SN-38 on colon adenocarcinoma cell lines expressing wild-type p53 (LS174T) or mutant non-functional p53 (HT29) have been investigated. On exposure to SN-38, HT29 cells rapidly progressed through G1 and S and arrested in G2/M. Release and concomitant increase in apoptosis after 48 h was concentration- and time-dependent (P < 0.001), being more rapid at higher concentrations, but reaching plateau at 10 ng ml–1 with prolonged exposure. LS174T cells showed only a small increase in apoptosis, and only at high concentrations (50–100 ng ml–1). The main effect of SN-38 in LS174T cells was prolonged cell cycle arrest, which was independent of concentration. Arrest occurred in all phases of the cell cycle, with the distribution depending on concentration (P < 0.001) and not duration (P > 0.05). With increasing concentration, LS174T cells arrested in G2/M, S and G1. Cell cycle arrest was coincident with increased p53 expression in each phase of the cell cycle. Expression in G1 increased with time and concentration (P < 0.001, P = 0.01 respectively), whereas in S and G2/M p53 expression increased only with time (P < 0.001). Dose-dependent p53-associated G1 arrest, in the absence of DNA synthesis indicates an additional cytotoxic mechanism for SN-38, which requires higher concentrations than the S phase mechanism, and detection of which seems to involve p53. For incubations with the same ED (exposure × duration), apoptosis in HT29 cells was significantly higher for prolonged exposure to lower concentrations, whereas in LS174T cells there was a trend towards increased apoptosis with shorter exposures to higher concentrations, indicating a schedule effect of SN-38. Although expression of wild-type p53 leads to a more rapid induction of apoptosis, SN-38 cytotoxicity was generally greater in cells with mutant p53, as wild-type cells escaped apoptosis by p53 associated prolonged cell cycle arrest. Thus, pulsed schedules with higher doses may be more effective in cells expressing wild-type p53, whereas continued exposure with protracted schedules may be more active in cells expressing mutant p53. © 1999 Cancer Research Campaign

PH‐dependent uptake of irinotecan and its active metabolite, SN‐38, by intestinal cells

Irinotecan (CPT-11) and its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), are believed to be reabsorbed by intestinal cells and to enter the entero-hepatic circulation, but there is little information to date. Our objective was to investigate the intestinal transport of CPT-11 and SN-38 in correlation with their associated cytotoxicity. Using either isolated hamster intestinal epithelial cells or/and human colon carcinoma HT29 cells, the uptake rates of [14C]CPT-11 and [14C]SN-38, both as respective non-ionic lactone form at acidic pH and anionic carboxylate form at basic pH, were investigated by the rapid vacuum filtration technique. The effect of physiologic intestinal luminal pH (6.2–8.0) on the uptake rate and cytotoxicity of SN-38 were estimated by the above method and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, respectively. The lactone forms of CPT-11 and SN-38 were transported passively, while the respective carboxylate form was absorbed actively. Uptake rates of both lactones were significantly higher than those of their carboxylates. Under physiologic pH, the respective uptake rates of CPT-11 and SN-38 were pH sensitive and decreased significantly by around 65%, at pH greater than 6.8. Furthermore, with decreasing pH, a higher uptake rate of SN-38 into HT29 cells correlates with a greater cytotoxic effect (r = 0.987). CPT-11 and SN-38 have absorption characteristics of weakly basic drugs such as short-chain fatty acids, suggesting that alkalization of the intestinal lumen may be critical to reduce their reabsorption and associated side effects. Int. J. Cancer 83:491–496, 1999. © 1999 Wiley-Liss, Inc.

PH-dependent uptake of irinotecan and its active metabolite, SN-38, by intestinal cells.

Irinotecan (CPT-11) and its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), are believed to be reabsorbed by intestinal cells and to enter the entero-hepatic circulation, but there is little information to date. Our objective was to investigate the intestinal transport of CPT-11 and SN-38 in correlation with their associated cytotoxicity. Using either isolated hamster intestinal epithelial cells or/and human colon carcinoma HT29 cells, the uptake rates of [(14)C]CPT-11 and [(14)C]SN-38, both as respective non-ionic lactone form at acidic pH and anionic carboxylate form at basic pH, were investigated by the rapid vacuum filtration technique. The effect of physiologic intestinal luminal pH (6.2-8.0) on the uptake rate and cytotoxicity of SN-38 were estimated by the above method and the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, respectively. The lactone forms of CPT-11 and SN-38 were transported passively, while the respective carboxylate form was absorbed actively. Uptake rates of both lactones were significantly higher than those of their carboxylates. Under physiologic pH, the respective uptake rates of CPT-11 and SN-38 were pH sensitive and decreased significantly by around 65%, at pH greater than 6.8. Furthermore, with decreasing pH, a higher uptake rate of SN-38 into HT29 cells correlates with a greater cytotoxic effect (r = 0.987). CPT-11 and SN-38 have absorption characteristics of weakly basic drugs such as short-chain fatty acids, suggesting that alkalization of the intestinal lumen may be critical to reduce their reabsorption and associated side effects.

Hyperthermic modulation of SN-38-induced topoisomerase I DNA cross-linking and SN-38 cytotoxicity through altered topoisomerase I activity.

The effect of different temperatures (37-42.5 degrees C) on SN-38 (the active metabolite of CPT-11) cytotoxicity was examined in the human lung carcinoma cell lines H460 and Calu-6 as well as the murine fibrosarcoma cell line L929. The cytotoxicity of SN-38, determined by MTT cell survival assay, was significantly increased in each cell line in combination with 41.8 degrees C hyperthermia (x60-120 min); the combination of SN-38 with 40.5 degrees C and 42.5 degrees C, however, was unchanged compared to 37 degrees C. Determination of topoisomerase (Topo) I DNA cross-linking in Calu-6 cells and L929 cells after treatment with SN-38 showed the same temperature profile as seen in the cell-survival assays with increased Topo I DNA cross-linking after treatment with the combination of SN-38 and 41.8 degrees C hyperthermia and unchanged Topo I DNA cross-linking at 40.5 degrees C and 42.5 degrees C. To test the hypothesis that increased Topo I DNA cross-linking and SN-38 cytotoxicity at 41.8 degrees C is caused by hyperthermia-modulated changes in Topo I activity, catalytic activity of Topo I extracted from each cell line and of purified human Topo I was determined at 20-42.5 degrees C. Topo I activity was found to be gradually increased with rising temperatures, resulting in significantly higher activity at 41.8 degrees C compared to 37 degrees C; further increase of temperature past 41.8 degrees C decreased Topo I activity back to levels found at 37 degrees C. Our data are used to explain a series of events resulting in hyperthermic enhancement of Topo I DNA cross-linking and SN-38 cytotoxicity in combination with 41.8 degrees C hyperthermia via increased Topo I activity.

Cytotoxicity of CPT-11 for gastrointestinal cancer cells cultured on fixed-contact-sensitive plates.

The cytotoxicity of SN-38, the major metabolite of CPT-11 (7-ethyl-10-[4-(1-piperidino)-1- piperidino]carbonyloxycamptothecin, was compared among gastrointestinal carcinomas of every organ, and between primary and metastatic lesions of every organ-originated gastrointestinal carcinoma, by an in vitro anticancer drug sensitivity test using fixed-contact-sensitive plates. The rates of cases having a high response (percent survival 75% or lower) to SN-38 but a low response (percent survival above 75%) to cisplatin, mitomycin C (MMC), adriamycin (ADM) and 5-fluorouracil (5-FU) were 14.6, 19.4, 15.6 and 27.0%, respectively. While, the rates of cases having a high response to cisplatin, MMC, ADM and 5-FU but a low response to SN-38 were 7.3, 2.8, 9.4 and 13.5%, respectively. Each of the former rates were higher than each of the latter rates. In particular, the former rate for MMC was significantly higher than the latter rate (p = 0.04). Two cases with colon cancer showed a high response only to SN-38. The percent survival of primary lesions in colon cancer was significantly lower than that in stomach cancer. The rates of hepatocellular carcinoma cases having a high response to SN-38 but a low response to cisplatin, MMC, ADM and 5-FU were 16.7, 16.7, 0 and 25%, respectively. Only one case had a high response to 5-FU but a low response to SN-38. The percent survival of metastatic lesions in pancreatic cancer was significantly lower than that of primary lesions. From this study, we recommend the further clinical trial of CPT-11 for colon and hepato-cellular cancers.

BCL-2 GENE PREVENTS INDUCTION OF APOPTOSIS IN L1210 MURINE LEUKEMIA-CELLS BY SN-38, A METABOLITE OF THE CAMPTOTHECIN DERIVATIVE CPT-11

New camptothecin (CPT) derivatives have recently been synthesized following the finding that CPT has strong antitumor activity due to its inhibition of topoisomerase I through the formation of stable topoisomerase I-DNA cleavable complexes, but has not been clinically used due to its pronounced toxicity. 7-ethyl-10-hydroxy-CPT (SN-38), a metabolite of the CPT derivative 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy-CPT(CPT-11), plays an essential role in mediating the antitumor effect of CPT-11. However, the reasons for the cytotoxicity of SN-38 remain unclear. In this study, we demonstrated using results of DNA fragmentation assay and cell cycle analysis that SN-38 and CPT both induce apoptosis in L1210 murine leukemia cells. We demonstrated in addition that enforced expression of the bcl-2 gene in L1210 cells by MPZenNeo (bcl-2) retroviral gene transfer increased resistance to the apoptosis induced by SN-38 and CPT. These findings suggest the possibility that the bcl-2 gene impedes the activity of a common pathway for apoptosis induced by SN-38 and CPT.