Regression of human colon cancer xenografts in SCID mice following oral administration of water-insoluble camptothecins

Gastrointestinal cancers pose major public health problems worldwide, in part because little progress has been made in the treatment of colorectal cancers. The present study explored the potential use of natural product topoisomerase I inhibitors, 10-hydroxycamptothecin (HCPT) and camptothecin (CPT), in the treatment of human colon cancers. HCPT and CPT are indole alkaloids originally isolated from th. Chinese tree, Camptotheca acuminata. They have been shown to have a wide spectrum of anticancer activity both in vitro and in vivo. The use of these camptothecins, however, has been hampered by their water-insolubility. In the present study, following screening of their in vitro antitumor activity, we determined their in vivo antitumor effects using C.B-17-scid/scid mice bearing LS174T or DLD-1 xenografts. Tumor-bearing mice were treated with oral doses of HCPT (1, 3, or 6 mg/kg/day) or CPT (1 or 3 mg/kg/day), 5 days per week for 2 weeks (LS 174T) or 3 weeks (DLD-1). Growth of the xenografts was significantly inhibited by HCPT and CPT in a dose-dependent manner, with marked reductions in tumor mass occurring in those groups given HCPT at 6 mg/kg/day or CPT at 3 mg/kg/day. Pathologic examination confirmed the dose-dependent atrophy of the adenocarcinomas. In summary, this study demonstrates the potential use of water-insoluble camptothecins when given by the oral route for treatment of human colon cancer, and provides the basis for the design of future human trials with these anticancer drugs.     

In vivo antitumor activity of bis(4,7-dimethyl-1,10-phenanthroline) sulfatooxovanadium(IV) (METVAN [VO(SO4)(Me2-Phen)2]).

The compound bis(4,7-dimethyl-1,10-phenanthroline) sulfatooxovanadium(IV) (METVAN [VO(SO4)(Me2-Phen)2]), exhibits potent cytotoxicity against human cancer cells at low micromolar concentrations. At concentrations > or = 1 microM, METVAN treatment was associated with a nearly complete loss of the adhesive, migratory, and invasive properties of the treated tumor cell populations. METVAN did not cause acute or subacute toxicity in mice at dose levels ranging from 12.5 mg/kg to 100 mg/kg. Therapeutic plasma concentrations > or = 5 microM were rapidly achieved and maintained in mice for at least 24 h after i.p. bolus injection of a single 10 mg/kg nontoxic dose of METVAN. At this dose level, the maximum plasma METVAN concentration was 37.0 microM, which was achieved with a t(max) of 21.4 min. Plasma samples (diluted 1:16) from METVAN-treated mice killed 85% of human breast cancer cells in vitro. METVAN was slowly eliminated with an apparent plasma t(1/2) of 17.5 h and systemic clearance of 42.1 ml/h/kg. In accordance with its potent in vitro activity and favorable in vivo pharmacokinetics, METVAN exhibited significant antitumor activity and delayed tumor progression in CB.17 severe combined immunodeficient (SCID) mouse xenograft models of human glioblastoma and breast cancer. In these experiments, METVAN was administered in daily injections of a single nontoxic 10 mg/kg i.p. dose on 5 consecutive days per week for 4 consecutive weeks beginning the day after the s.c. inoculation of U87 glioblastoma or MDA-MB-231 breast cancer cells. At 40 days after the inoculation of tumor cells, the U87 tumor xenografts in the vehicle-treated control SCID mice were much larger than those of the mice treated with METVAN (4560 +/- 654 mm(3) versus 1688 +/- 571 mm(3); P = 0.003). Similarly, the MDA-MB-231 tumors in SCID mice treated with METVAN were much smaller 40 days after tumor cell inoculation than those of the vehicle-treated control SCID mice (174 +/- 29 mm(3) versus 487 +/- 82 mm(3); P = 0.002). The favorable in vivo pharmacodynamic features and antitumor activity of METVAN warrants further development of this novel oxovanadium compound as a potential new anticancer agent.     

Potent combination therapy for human breast tumors with high doses of 5-fluorouracil: remission and lack of host toxicity

Purpose

The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in reducing 5-fluorouracil (FUra) host toxicity and enhancing its chemotherapeutic efficacy against human breast tumors. PTAU is a potent and specific inhibitor of uridine phosphorylase (UP, EC 2.4.2.3), the enzyme responsible for uridine catabolism.

Methods

SCID mice bearing MDA-MB-468 and MCF-7 human breast tumors were injected intraperitoneally with FUra (50, 200 or 300?mg/kg) on days 17, 24, and 31 after tumor cell inoculation. PTAU (120?mg/kg), uridine (1,320?mg/kg), or their combination was administered orally two or 4?h after FUra injection. Another four administrations of PTAU plus uridine were given every 8?h after the first treatment with PTAU plus uridine. Survival and body weight were used to evaluate host toxicity. Tumor weight was used to evaluate the efficacy of the drugs on tumor growth. The mice were monitored for 38?days.

Results

Administration of the maximum tolerated dose (50?mg/kg) of 5-fluorouracil (FUra) to SCID mice bearing human breast MDA-MB-468 and MCF-7 adenocarcinoma tumor xenografts reduced tumor weight by 59 and 61%, respectively. Administration of 200?mg/kg FUra resulted in 100% mortality. Oral administration of uridine (1,320?mg/kg) alone, 2?h following the administration of 200?mg/kg FUra, did not rescue from FUra host toxicity as all the mice died. Administration of 120?mg/kg PTAU resulted in partial rescue from this lethal dose of FUra as 38% of inoculated mice survived and the tumor weights were reduced by approximately 67%. Coadministration of PTAU plus uridine resulted in complete rescue from the toxicity of FUra. All of the mice survived, and MDA-MB-468 and MCF-7 tumor weights were reduced by 97% and total remission, respectively. Doubling the FUra treatment dose to 400?mg/kg in the MDA-MB-468 inoculated mice, with the administration of the adjuvant combination treatment of PTAU plus uridine, was unsuccessful in rescuing from FUra toxicity as all the mice died. Lowering the dose of FUra to 300?mg/kg, under the same conditions, resulted in 67% mice survival, and the MCF-7 tumor weights were reduced by 100%. Treatment with uridine alone did not protect from FUra toxicity at 200, 300, and 400?mg/kg as all of the mice died. At the higher dose of 300 and 400?mg/kg FUra, PTAU alone had no rescuing effect. There was no significant difference between MDA-MB-468 and MCF-7 in their response to the different regimens employed in this study in spite of the fact that MDA-MB-468 is estrogen receptor negative while MCF-7 is estrogen receptor positive.

Conclusions

The present results demonstrate that the combination of PTAU plus uridine represents an exceptionally efficient method in increasing FUra chemotherapeutic efficacy while minimizing its host toxicity. The efficiency of the PTAU plus uridine combination can be attributed to the extraordinary effectiveness of this combination treatment in raising and maintaining higher levels of uridine in vivo (Al Safarjalani et al. in Cancer Chemo Pharmacol 55:541–551, 2005). Therefore, the combination of PTAU plus uridine can provide a better substitute for the massive doses of uridine necessary to rescue or protect from FUra host-toxicities, without the toxic side effects associated with such doses of uridine. The combination may also allow the escalation of FUra doses for better chemotherapeutic efficacy against human breast carcinoma, with the possibility of avoiding FUra host-toxicities. Alternatively, the combination of PTAU and uridine may be useful as an antidote in the few cases when cancer patients receive a lethal overdose of FUra.     

开环及闭环羟基喜树碱对口腔鳞癌细胞株抗癌作用的研究

背景与目的:羟基喜树碱(hydroxycamptothecin,HCPT)的抗癌活性与其内酯环(包括闭环和开环)密切相关,但关于闭环HCPT和开环HCPT的抗癌活性仍有争议,研究已显示开环HCPT对口腔鳞癌具有明显的体内外抑制作用,本文比较开环及闭环HCPT对口腔鳞癌细胞株Tca8113的体内外抗瘤作用及其作用机理。方法:采用开环及闭环HCPT处理Tca8113细胞及其裸鼠移植瘤,MTT法及流式细胞仪(FCM)检测HCPT对Tca8113细胞的细胞毒作用及对细胞周期的影响;观察经HCPT处理后Tca8113细胞移植瘤的生长状态,计算肿瘤倍增时间和抑瘤率;高效液相色谱仪检测裸鼠血浆及移植瘤中HCPT的浓度,计算药代动力学参数。结果:开环及闭环HCPT对Tca8113细胞具有相同的强杀伤作用,FCM检测显示小于1μmol/LHCPT可将细胞阻滞在S期和G2/M期,100μmol/LHCPT则诱导细胞凋亡。与对照组比较,HCPT组移植瘤生长减慢,倍增时间延长,肿瘤抑制率分别为69.6%(3mg/kg开环HCPT)、65.0%(3mg/kg闭环HCPT)和74.1%(10mg/kg开环HCPT)。腹腔注射10mg/kgHCPT后裸鼠血浆曲线下面积分别为2.66μg·h·ml-1(闭环HCPT)和0.42μg·h·ml-1(开环HCPT),肿瘤组织中可检测到HCPT。HCPT3mg/kg组未见明显的毒副作用,开环HCPT10mg/kg组可见明显的胃肠道反应,闭环HCPT10mg/kg组所有裸鼠死亡。结论:开环及闭环HCPT均对口腔鳞癌细胞具有强的体内外细胞毒作用,其机理与阻断细胞于S期和G2/M期以及诱导细胞凋亡有关,但闭环HCPT毒性较开环HCPT毒性大。     

Radiosensitization by antisense anti-MDM2 mixed-backbone oligonucleotide in in vitro and in vivo human cancer models.

PURPOSE: The MDM2 oncogene, amplified or overexpressed in many human cancers, has been suggested to be a novel target for cancer therapy. We have demonstrated a second-generation antisense antihuman-MDM2 oligonucleotide to have antitumor activity when administered alone or in combination with cancer chemotherapeutic agents. In the present study, we investigated the effect of the antisense oligonucleotide on radiation therapy. EXPERIMENTAL DESIGN: The in vitro radiosensitization activity was determined in cell lines of human cancers of prostate (LNCaP and PC3), breast (MCF-7 and MDA-MB-468), pancreas (PANC-1), and glioma (U87-MG and A172) and its in vivo radiosensitization activity in xenograft models of LNCaP, PC3, MCF-7, MDA-MB-468, and PANC-1. RESULTS: In cells containing at least one functional p53 allele (LNCaP, U87-MG, and A172), after specific inhibition of MDM2 expression, p53 and p21 levels were elevated. In LNCaP cells, the Bax level was increased, and Bcl-2 and E2F1 levels were decreased. In PC3 cells that are p53 null, after inhibition of MDM2 expression, Bax and p21 levels were elevated, and E2F1 levels were decreased. On the basis of in vitro clonogenic assay, the antisense oligonucleotide, in a sequence-specific manner, significantly increased radiation-induced antiproliferation effects. It also increased radiation-induced inhibitory effects on tumor growth in SCID or nude mice bearing LNCaP, PC3, MCF-7, MDA-MB-468, and PANC-1 xenografts. CONCLUSIONS: These results suggest that MDM2 has a role in radiation therapy of human cancers, regardless of p53 status, providing a basis for future development of MDM2 inhibitors, such as antisense oligonucleotides, as radiosensitizers.     

羟基喜树碱纳米微球抗肿瘤作用的实验研究

目的：研究载羟基喜树碱（Hydroxycamptothecin，HCPT）的聚乙二醇-聚谷氨酸苄酯（PEG—PBLG）纳米微球对口腔鳞癌及结肠癌的体内抗瘤作用。方法：采用超透析法制备HCPT／PEG—PBLG纳米胶束，透射电镜观察纳米微球的形态．应用HCPT／PEG—PBLG纳米微球治疗口腔鳞癌或结肠癌裸鼠移植瘤，观察移植瘤的生长状态及组织病理学改变。结果：HCPT／PEG—PBLG纳米微球为核-壳型结构，大小约230nm；于对照组比较，所有HCPT治疗组均可明显抑制口腔鳞癌或结肠癌移植瘤的生长（P〈0．01），其中HCPT／PEG—PBLG纳米微球组的抑瘤率明显高于HCPT组（P〈0．01）．PEG—PBLG组对移植瘤的生长无抑制作用（P〉0．05）；对口腔鳞癌移植瘤的抑瘤率分别为69．6％（HCPT，3mg／kg，qd）、74．1％（HCPT，10mg／kg，qd）、59．8％（HCPT，3mg／kg，qod）和85．6％（HCPT／PEG—PBLG3mg／kg，qod）；对结肠癌移植瘤的抑瘤率分别为70．0％（HCPT，3mg／kg，qod）和83．8％（HCPT／PEG—PBLG3mg／kg，qod）。组织病理学显示：PEG—PBLG、HCPT／PEG—PBLG组肿瘤细胞可见较多吞噬小体，而HCPT组未见；HCPT、HCPT／PEG—PBLG组肿瘤细胞可见较多凋亡和坏死。结论：HCPT／PEG—PBLG纳米微球可明显提高HCPT的抑瘤作用，PEG—PBLG纳米微球可成为HCPT类药物的理想新型载体。     

Efficacy, pharmacokinetics, tisssue distribution, and metabolism of the Myc–Max disruptor, 10058-F4 [Z,E]-5-[4-ethylbenzylidine]-2-thioxothiazolidin-4-one, in mice

Objectives  c-Myc is commonly activated in many human tumors and is functionally important in cellular proliferation, differentiation, apoptosis and cell cycle progression. The activity of c-Myc requires noncovalent interaction with its client protein Max. In vitro studies indicate the thioxothiazolidinone, 10058-F4, inhibits c-Myc/Max dimerization. In this study, we report the efficacy, pharmacokinetics and metabolism of this novel protein–protein disruptor in mice. Methods  SCID mice bearing DU145 or PC-3 human prostate cancer xenografts were treated with either 20 or 30 mg/kg 10058-F4 on a qdx5 schedule for 2 weeks for efficacy studies. For pharmacokinetics and metabolism studies, mice bearing PC-3 or DU145 xenografts were treated with 20 mg/kg of 10058-F4 i.v. Plasma and tissues were collected 5–1440 min after dosing. The concentration of 10058-F4 in plasma and tissues was determined by HPLC, and metabolites were characterized by LC-MS/MS. Results  Following a single iv dose, peak plasma 10058-F4 concentrations of approximately 300 μM were seen at 5 min and declined to below the detection limit at 360 min. Plasma concentration versus time data were best approximated by a two-compartment, open, linear model. The highest tissue concentrations of 10058-F4 were found in fat, lung, liver, and kidney. Peak tumor concentrations of 10058-F4 were at least tenfold lower than peak plasma concentrations. Eight metabolites of 10058-F4 were identified in plasma, liver, and kidney. The terminal half-life of 10058-F4 was approximately 1 h, and the volume of distribution was >200 ml/kg. No significant inhibition of tumor growth was seen after i.v. treatment of mice with either 20 or 30 mg/kg 10058-F4. Conclusion  The lack of significant antitumor activity of 10058-F4 in tumor-bearing mice may have resulted from its rapid metabolism and low concentration in tumors.     

Anti-metastasis Activity of Black Rice Anthocyanins Against Breast Cancer: Analyses Using an ErbB2 Positive Breast Cancer Cell Line and Tumoral Xenograft Model

Background: Increasing evidence from animal, epidemiological and clinical investigations suggest that dietary anthocyanins have potential to prevent chronic diseases, including cancers. It is also noteworthy that human epidermal growth factor receptor 2 (ErbB2) protein overexpression or ErbB2 gene amplification has been included as an indicator for metastasis and higher risk of recurrence for breast cancer. Materials and Methods: The present experiments investigated the anti-metastasis effects of black rice anthocyanins (BRACs) on ErbB2 positive breast cancer cells in vivo and in vitro. Results: Oral administration of BRACs (150 mg/kg/day) reduced transplanted tumor growth, inhibited pulmonary metastasis, and decreased lung tumor nodules in BALB/c nude mice bearing ErbB2 positive breast cancer cell MDA-MB-453 xenografts. The capacity for migration, adhesion,motility and invasion was also inhibited by BRACs in MDA-MB-453 cells in a concentration dependent manner, accompanied by decreased activity of a transfer promoting factor, urokinase-type plasminogen activator (u-PA). Conclusions: Together, our results indicated that BRACs possess anti-metastasis potential against ErbB2 positive human breast cancer cells in vivo and in vitro through inhibition of metastasis promoting molecules.     

MEK1/2 inhibition promotes Taxotere lethality in mammary tumors in vivo

Taxol (paclitaxel) and Taxotere (docetaxel) are considered as two of the most important anti-cancer chemotherapy drugs. The cytotoxic action of these drugs has been linked to their ability to inhibit microtubule depolymerization, causing growth arrest and subsequent cell death. Studies by a number of laboratories have also linked suppression of MEK1/2 signaling to enhanced Taxol toxicity in vitro and in vivo. The present study examined the interactions of the semi-synthetic taxane Taxotere with MEK1/2 inhibitors in epithelial tumor cells. In vitro colony formation studies demonstrated that Taxotere and the MEK1/2 inhibitor PD184352 interacted in a sequence dependent fashion to synergistically kill human mammary carcinoma cells (MDA-MB-231, MCF7) as well as in other tumor cell types; e.g. prostate and renal cell carcinoma. Athymic mice were implanted in the rear flank with either MDA-MB-231 or MCF7 cells and tumors permitted to form to a volume of approximately 100 mm3 prior to a two day exposure of either Vehicle, PD184352 (25 mg/kg), Taxotere (15 mg/kg) or the drug combination. Tumor volume was measured every other day and tumor growth determined over the following approximately 30 days. Transient exposure of MDA-MB-231 tumors or MCF7 tumors to PD184352 did not significantly alter tumor growth rate or the mean tumor volume in vivo approximately 15-30 days after drug administration. Transient Taxotere exposure of MDA-MB-231 or to a lesser extent MCF7, tumors modestly reduced the mean tumor volume in vivo approximately 15-30 days after drug administration. In contrast, combined treatment with PD184352 and Taxotere significantly reduced MDA-MB-231 and MCF7 tumor growth. The tumor control values for MDA-MB-231 cells and MCF7 cells were 0.43 and 0.71, respectively. Fractionated irradiation of MDA-MB-231 tumors during drug exposure or single dose irradiation prior to drug administration did not significantly further suppress tumor growth beyond that of cells exposed to Taxotere and MEK1/2 inhibitor. Single dose irradiation of tumors after drug exposure, however, caused a significant further suppression of tumor growth below that caused by drug exposure. These findings were also reflected in ex vivo colony formation analyses of isolated tumor cells. Collectively, these findings argue that Taxotere and MEK1/2 inhibitors have the potential to suppress mammary tumor growth in vivo which is enhanced by sequence-dependent exposure to ionizing radiation. Based on the cell lines used in these studies, our findings argue that the interaction of Taxotere and PD184352 is independent of p53 status, estrogen dependency, caspase 3 levels or oncogenic K-RAS expression.     

Selective modulation of the therapeutic efficacy of anticancer drugs by selenium containing compounds against human tumor xenografts.

PURPOSE: Studies were carried out in athymic nude mice bearing human squamous cell carcinoma of the head and neck (FaDu and A253) and colon carcinoma (HCT-8 and HT-29) xenografts to evaluate the potential role of selenium-containing compounds as selective modulators of the toxicity and antitumor activity of selected anticancer drugs with particular emphasis on irinotecan, a topoisomerase I poison. EXPERIMENTAL DESIGN: Antitumor activity and toxicity were evaluated using nontoxic doses (0.2 mg/mouse/day) and schedule (14-28 days) of the selenium-containing compounds, 5-methylselenocysteine and seleno-L-methionine, administered orally to nude mice daily for 7 days before i.v. administration of anticancer drugs, with continued selenium treatment for 7-21 days, depending on anticancer drugs under evaluation. Several doses of anticancer drugs were used, including the maximum tolerated dose (MTD) and toxic doses. Although many chemotherapeutic agents were evaluated for toxicity protection by selenium, data on antitumor activity were primarily obtained using the MTD, 2 x MTD, and 3 x MTD of weekly x4 schedule of irinotecan. RESULTS: Selenium was highly protective against toxicity induced by a variety of chemotherapeutic agents. Furthermore, selenium increased significantly the cure rate of xenografts bearing human tumors that are sensitive (HCT-8 and FaDu) and resistant (HT-29 and A253) to irinotecan. The high cure rate (100%) was achieved in nude mice bearing HCT-8 and FaDu xenografts treated with the MTD of irinotecan (100 mg/kg/week x 4) when combined with selenium. Administration of higher doses of irinotecan (200 and 300 mg/kg/week x 4) was required to achieve high cure rate for HT-29 and A253 xenografts. Administration of these higher doses was possible due to selective protection of normal tissues by selenium. Thus, the use of selenium as selective modulator of the therapeutic efficacy of anticancer drugs is new and novel. CONCLUSIONS: We demonstrated that selenium is a highly effective modulator of the therapeutic efficacy and selectivity of anticancer drugs in nude mice bearing human tumor xenografts of colon carcinoma and squamous cell carcinoma of the head and neck. The observed in vivo synergic interaction is highly dependent on the schedule of selenium.     

The antitumor activity of NK012, an SN‐38–incorporating micelle,in combination with bevacizumab against lung cancer xenografts

BACKGROUND:

It has been demonstrated that NK012, a novel 7‐ethyl‐10‐hydroxycamptothecin (SN‐38)‐incorporating polymeric micelle, exerts significantly more potent antitumor activity against various human tumor xenografts than irinotecan (CPT‐11) (a water‐soluble prodrug of SN‐38). Combination therapy of anticancer agents with bevacizumab (Bv), an anti‐vascualr endothelial growth factor humanized monoclonal antibody, has more potently inhibited tumor growth than either agent alone. In the current study, the authors examined the antitumor effect of NK012 in combination with Bv against human lung cancer.

METHODS:

Nude mice bearing lung adenocarcinoma (PC‐14 or A549 xenografts) were administered NK012 at SN‐38‐equivalent doses of 5 mg/kg or 30 mg/kg in combination with or without Bv at 5 mg/kg. CPT‐11 at a dose of 66.7 mg/kg was administered with or without Bv at a dose of 5 mg/kg in the same experimental model. To evaluate interaction with Bv, the pharmacokinetics and microvessel density in tumors that were treated on each regimen were analyzed.

RESULT:

In vitro, the growth‐inhibitory effect of NK012 was 50‐fold more potent than that of CPT‐11 and was almost equivalent to that of SN‐38. In vivo studies revealed that the combination of NK012 plus Bv had significantly greater antitumor activity against human lung cancer xenografts compared with NK012 alone (PC‐14, P = .0261; A549, P < .001). The pharmacokinetic profile of NK012 revealed that coadministration of Bv did not interfere with the accumulation of NK012.

CONCLUSIONS:

In this study, significant antitumor activity was noted with NK012 in combination with Bv against lung cancer cells. The current results warrant the clinical evaluation of NK012 in lung cancer. Cancer 2010. © 2010 American Cancer Society.     

羟基喜树碱和鬼臼乙叉甙联合治疗鼻咽癌的实验研究

背景与目的:许多研究表明,拓扑异构酶Ⅰ和Ⅱ抑制剂具有互补作用,两者合用有协同作用。本研究探讨拓扑异构酶Ⅰ和Ⅱ抑制剂犤羟基喜树碱(HCPT)和鬼臼乙叉甙(VP-16)犦联合治疗鼻咽癌实验研究。方法:MTT法测定HCPT和VP-16单独及联合应用对鼻咽癌细胞株CNE2的细胞毒作用。建立CNE2裸鼠移植瘤模型,观察HCPT和VP-16联合应用体内抑制肿瘤生长的作用。结果:HCPT和VP-16单独应用对CNE2具有明显的细胞毒作用,其IC50分别为(13.5±1.2)μmol/L及(13.5±1.0)μmol/L。在体外实验中,在HCPT与VP-16相同剂量条件下,高浓度合用具有明显的协同作用(CI<1)。体内实验也表明,1.5mg/kg每2天1次,HCPT抑瘤率为13.6%;10mg/kg每2天1次,VP-16抑瘤率为27.3%;VP-16与HCPT合用,其抑瘤率为50%。结论:HCPT+VP-16在体内、外可协同抑制鼻咽癌细胞的生长。     

Growth inhibition and antimetastatic effect of antisense poly-DNP-RNA on human breast cancer cells

The RNase-resistant and membrane-permeable antisense poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotides (poly-DNP-RNA) against RIalpha subunit of protein kinase A (RIalpha/PKA) has been used to inhibit the growth of human breast cancer MDA-MB-231 cells in vitro and in vivo. This antisense poly-DNP-RNA, with oligonucleotide sequence GGGCGUGCCUCCUCACUGGC, was found to be an effective concentration-dependent inhibitor of MDA-MB-231 cell line, whereas the control poly-DNP-RNAs with either random or sense sequence were found completely inactive. In situ hybridization studies showed that this antisense inhibitor can permeate spontaneously into MDA-MB-231 cells and distribute itself throughout the cytoplasm. Intraperitoneal administration of this antisense RIalpha poly-DNP-RNA to SCID mice with transplanted MDA-MB-231 cells was found to inhibit the growth of the xenografts in a concentration-dependent way, prevent metastasis, and drastically reduce mortality.     

Synergism between the anticancer actions of 2-methoxyestradiol and microtubule-disrupting agents in human breast cancer

2-Methoxyestradiol (2-MeO-E(2)), a well-known nonpolar endogenous metabolite of 17beta-estradiol, has strong antiproliferative, apoptotic, and antiangiogenic actions in vitro and in vivo at pharmacologic concentrations. We determined in the present study whether 2-MeO-E(2) can enhance the anticancer actions of paclitaxel or vinorelbine (two commonly used microtubule-disrupting agents) in several human breast cancer cell lines, including the estrogen receptor-positive MCF-7 and T-47D cells and the receptor-negative MDA-MB-435s and MDA-MB-231 cells. 2-MeO-E(2) in combination with paclitaxel or vinorelbine exhibited a synergistic anticancer effect in these human breast cancer cells in vitro, and this synergistic effect was more pronounced when each of the drugs was used at relatively low concentrations. Additional experiments using female athymic BALB/c nu/nu mice showed that p.o. administration of 2-MeO-E(2) at 30 mg/kg body weight, once a week for 6 weeks, markedly enhanced the activity of paclitaxel or vinorelbine against the growth of the estrogen receptor-negative MDA-MB-231 human breast cancer xenografts in these animals. By contrast, combination of 2-MeO-E(2) with 5-fluorouracil only had a partial additive effect against the growth of these cell lines in culture, and no synergistic effect was observed. Interestingly, when doxorubicin was used in combination with 2-MeO-E(2), the antiproliferative effect of 2-MeO-E(2) was somewhat antagonized by doxorubicin when it was present at high concentrations. Our results showed that 2-MeO-E(2) at nontoxic or subtoxic doses selectively enhanced the effects of certain microtubule-disrupting agents (such as paclitaxel and vinorelbine) against the growth of the receptor-negative human breast cancer cells in culture and also in athymic nude mice.     

Development of a new isogenic cell-xenograft system for evaluation of NAD(P)H:quinone oxidoreductase-directed antitumor quinones: evaluation of the activity of RH1.

PURPOSE: The purpose of our study was to develop and validate an isogenic cell line pair that differs only in the expression of NAD(P)H:quinone oxidoreductase (NQO1) that can be used to examine the in vitro and in vivo role of NQO1 in the bioactivation of the antitumor quinone RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), a compound currently in Phase I clinical trials. EXPERIMENTAL DESIGN: MDA-MB-468 (MDA468) human breast adenocarcinoma cells, homozygous for a polymorphism in NQO1 (NQO1*2/*2) and with low levels of NQO1 activity, were stably transfected with human NQO1 to generate a clone (NQ16) expressing very high NQO1 activity. We examined levels of other reductases and looked at biochemical systems that might influence response to antitumor quinones to validate that the isogenic cell line pair differed only in the expression of NQO1. The 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium (MTT) assay was used to determine the differential toxicity of various quinones, including the most recent NQO1-directed antitumor quinone, RH1, between the two cell lines. Human tumor xenografts were established from both MDA468 and NQ16 cells, and the antitumor activity of RH1 was evaluated. RESULTS: Levels of cytochrome P450 reductase, cytochrome b(5) reductase, soluble thiols, and superoxide dismutase in the NQ16 line were unchanged from the parental line. The functional significance of wild-type NQO1 expression was confirmed by measurement of the differential toxicity of compounds activated or deactivated by NQO1 in the two cell lines. The toxicity of the NQO1-directed antitumor quinones RH1 and streptonigrin were markedly greater and the toxicity of menadione, which is detoxified by NQO1, was ameliorated in the NQ16 line. High levels of NQO1 expression were observed throughout xenograft tumors established from the NQ16 cell line. RH1 treatment was effective at statistically reducing tumor volume in NQ16 xenografts at all of the doses tested (0.1, 0.2, 0.4 mg/kg every day for 5 days), whereas only the highest dose of RH1 resulted in a significant reduction in tumor volume in MDA468 xenografts. CONCLUSIONS: The MDA468/NQ16 isogenic cell line pair is a useful model system for evaluating the role of NQO1 in the bioactivation of antitumor quinones in both cell lines and xenografts. In addition, our data demonstrate that the novel antitumor quinone RH1, is effectively activated by NQO1 both in vitro and in vivo.     

Thermographic assessment of tumor growth in mouse xenografts

In human breast tumors, a 1-2 degrees C increase in skin surface temperature is usually observed at the periphery; it has been proposed that this change is due to the hypervascularity and increased blood flow resulting from tumor-associated angiogenesis. Here we tested the hypothesis that thermal imaging might represent a useful adjunctive technique in monitoring the growth dynamics of human tumor xenografts. Xenografts were established in immunocomprised nude mice using MDA-MB-231 or MCF7 breast cancer cells. We exploited the inherent noncontact and noninvasive advantages of infrared thermography to detect skin surface temperature changes. Continuous thermographic investigation was performed to detect and monitor tumor growth in vivo and high resolution digital images were analyzed to measure the tumor temperature dynamics. In contrast to the skin temperature increases associated with human breast cancer, a consistent temperature decrease was found in the xenograft mice. In one case, a smaller secondary tumor, otherwise undetectable, was clearly evident by thermal imaging. The tumors were cooler than the surrounding tissue with a maximum temperature reduction of 1.5 degrees C for MDA-MB-231 tumor and 3 degrees C for MCF7 tumors observed on day 14. In addition, the temperature of the xenograft tumors decreased progressively as they grew throughout the observation period. It was demonstrated that thermographic imaging could detect temperature changes as small as 0.1 degrees C on the skin surface at an early stage of tumor development. The findings of the study indicate that thermographic imaging might have considerable potential in monitoring human tumor xenografts and their response to anticancer drugs.     

Inhibition of growth of human nasopharyngeal cancer xenografts in SCID mice by arsenic trioxide

AIMS AND BACKGROUND: It is known that arsenic trioxide (As2O3) can induce clinical remission in patients suffering from acute promyelocytic leukemia. It has been suggested that the agent might also be effective against other malignancies. This study was done to explore the efficacy of As2O3 in the treatment of human nasopharyngeal cancer xenografts in SCID (severe combined immunodeficiency) mice. METHODS: Human nasopharyngeal cancer cells from the CSNE-1 cell line were implanted subcutaneously into SCID mice to produce tumors. The tumor inhibitory rate in vivo was assessed after intraperitoneal administration of As2O3. Histopathological changes in the tumor tissues and the toxicity of As2O3 to the liver, heart and kidneys of the host mice were also investigated. RESULTS: At doses of 1 mg/kg and 5 mg/kg As2O3 induced apoptosis in nasopharyngeal carcinoma cells. At 5 mg/kg As2O3 also induced cancer cell differentiation, it reduced the PCNA expression, and inhibited tumor growth. The tumor growth inhibitory rate in this experimental group was 76.02%. No nephrotoxicity was observed histologically at these dose levels but some pathological changes in liver and cardiac tissues were found. As2O3 proved lethal to the SCID mice at a dose of 10 mg/kg. CONCLUSION: As2O3 has an inhibitory effect on human nasopharyngeal carcinoma xenografts in SCID mice. The mechanism of antitumor activity may be due, at least in part, to the induction of apoptosis and differentiation in cancer cells.     

羟基喜树碱对口腔鳞癌细胞株的抗癌作用

背景与目的:研究已显示羟基喜树碱(hydroxycamptothecin,HCPT)对多种恶性肿瘤具有良好的治疗效果,但对口腔鳞癌的治疗研究还很少,本文研究羟基喜树碱对口腔鳞癌细胞株(Tca8113细胞株)的体内外抗瘤作用及其作用机制。方法:采用MTT法及FCM(flowcytometry)检测HCPT对Tca8113细胞株的体外细胞毒作用及对细胞周期的影响;Tca8113细胞株裸鼠移植瘤经HCPT处理后观察肿瘤的生长状态,计算倍增时间和抑瘤率。结果:HCPT对Tca8113细胞具有强的杀伤作用,其IC50为2μmol/L;FCM检测显示低浓度HCPT处理后可将细胞先后阻滞在S期和G2+M期,高浓度的HCPT则诱导细胞凋亡,细胞凋亡率随药物浓度和作用时间延长明显上升;与对照组比较,HCPT组移植瘤生长减慢,倍增时间延长,两组间肿瘤体积存在显著性差异(P<0.001),接种后28天其抑瘤率达69.6%。结论:HCPT对口腔鳞癌细胞具有强的细胞毒作用,对Tca8113移植瘤生长有显著抑制作用,其机制与阻断细胞周期及诱导细胞凋亡有关。     

Insulin like growth factor binding protein-7 reduces growth of human breast cancer cells and xenografted tumors

Previously, we have shown that insulin-like growth factor binding protein-7 (IGFBP-7) expression is inversely correlated with disease progression in breast cancer and is associated with poor outcome. To further investigate the role of IGFBP-7 in the growth and metastatic behavior of breast cancer, primary breast tumors and metastatic tumors derived from the same patients were analyzed for IGFBP-7 expression. Immunohistochemical analysis revealed that IGFBP-7 is downregulated in half of the human metastatic breast tumors tested. IGFBP-7 has been linked to suppression of oncogenic pathways and can directly restore cellular senescence in melanomas, leading to their regression. It is possible that breast tumors with metastatic potential have escaped from IGFBP-7-induced suppression by its down-regulation. Twenty-two human primary breast tumor specimens were transplanted into human-bone NOD/SCID mice. One of the two triple negative primary breast tumors was serially xenotransplanted more than five times. Each serial transplant resulted in increased tumor take and rate of growth. Expression of IGFBP-7 was downregulated upon each serial implantation. To investigate the role of IGFBP-7 in breast tumor suppression, IGFBP-7 was overexpressed in the triple negative MDA-MB-468 human breast cancer line by stable transfection of a pSec-tag2-IGFBP-7 vector. The parental MDA-MB-468 breast cancer cells expressed extremely low levels of endogenous IGFBP-7. The production of IGFBP-7 protein by the MDA-MB-468 cells stably transfected with IGFBP-7 was confirmed by immunoblotting with anti-IGFBP-7 antibody. Ectopic overexpression of IGFBP-7 significantly reduced the growth of the IGFBP-7 transfected MDA-MB-468 cells compared to the parental MDA-MB-468 cells. We also assessed the role of IGFBP-7 on cell migration, a key determinant of malignant progression and metastasis. When parental MDA-MB-468 cells were treated with various amounts of conditioned medium derived from the IGFBP-7 overexpressing cell line, a significant difference in cell migration rate was observed between untreated and treated cells. IGFBP-7 strongly suppressed the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK-1/2, suggesting that IGFBP-7 mediates its anti-proliferative effects through negative feedback signaling. Levels of phospho-ERK-1/2 were higher in the parental MDA-MB-468 than in IGFBP-7-expressing cells derived from it. When injected subcutaneously into NOD/SCID mice, the increased expression of IGFBP-7 in the MDA-MB-468 transfected cells reduced the rate of tumor growth in comparison to the parental MDA-MB-468 controls. These results suggest that the growth of breast cancer could be prevented by the forced expression of IGFBP-7 protein.