EMT6 Mouse Mammary Tumor Research Articles

Distribution of Selenium and Oxidative Stress in Breast Tumor-Bearing Mice

The present study investigated the effects of breast tumors on the blood and tissue distribution of essential trace mineral selenium (Se), and oxidative stress status of mice. Female 10-week-old BALB/cByJNarl mice were randomly assigned into control (CNL) and breast tumor-bearing (TB) groups. TB mice were injected subcutaneously into the right hind thigh with 5 × 106 EMT6 mouse mammary tumor cells. After 22 days, we measured Se concentrations, Se-dependent glutathione peroxidase (GPx) activities, and malondialdehyde (MDA) products (indicator of oxidative stress) in plasma, various tissues, and plasma vascular endothelial growth factor (VEGF) concentrations. There were no significant differences in body weights and daily intake between both groups. Compared with the CNL group, TB mice have decreases in plasma Se concentrations and GPx activities, as well as higher plasma VEGF and MDA concentrations. Plasma Se concentrations were also negatively correlated with plasma MDA and VEGF concentrations. Furthermore, tissue Se concentrations and GPx activities in TB animals were lower; whereas the MDA concentrations higher in various tissues including liver, kidney, brain, lung, spleen, and thymic tissues. In conclusion, disruption of Se homeostasis critically reflects oxidative stress in target tissues, thus may increase the risk for progression of breast cancer and metastasis.

Preclinical studies of the Chinese Herbal Medicine formulation PHY906 (KD018) as a potential adjunct to radiation therapy

Objective: Abdominal and pelvic radiotherapy is limited by the radiosensitivity of the small and large intestine. PHY906 (KD018), a state-of-the-art, well defined adaptation of a traditional Chinese medicine, decreased intestinal injury from chemotherapy in preclinical studies and is in clinical trials with chemotherapy. This project assessed whether PHY906 would also reduce intestinal injury from abdominal irradiation in mice.Materials and methods: BALB/c mice received whole-abdomen irradiation (2 Gy/day) ± PHY906 by oral gavage twice daily for 4 days. Intestinal injury was assayed by physiological observations and histological studies. Effects of PHY906 on EMT6 mouse mammary tumors were assayed in tumor growth studies.Results: PHY906 decreased toxicity from fractionated abdominal irradiation. Radiation alone produced marked blunting and loss of villi, crypt hyperplasia and irregular crypt morphology, which were reduced by PHY906. The radiation-induced reduction in viable crypt numbers was also mitigated by PHY906. PHY906 did not alter radiation-induced weight loss, but resulted in more rapid recovery. PHY906 did not alter tumor growth, local invasion or metastatic spread and did not protect tumors from growth delays produced by single-dose or fractionated irradiation.Conclusion: In this mouse model, PHY906 (KD018) decreased the toxicity of abdominal irradiation without protecting tumors and thereby increased the therapeutic ratio.

Preliminary Studies with a New Hypoxia-Selective Cytotoxin, KS119W,In VitroandIn Vivo

Agents with selective toxicity to hypoxic cells have shown promise as adjuncts to radiotherapy. Our previous studies showed that the bioreductive alkylating agent KS119 had an extremely large differential toxicity to severely hypoxic and aerobic cells in cell culture, and was effective in killing the hypoxic cells of EMT6 mouse mammary tumors in vivo. However, the limited solubility of that compound precluded its development as an anticancer drug. Here we report our initial studies with KS119W, a water-soluble analog of KS119. The cytotoxicity of KS119W to EMT6 cells in vitro was similar to that of KS119, with both agents producing only minimal cytotoxicity to aerobic cells even after intensive treatments, while producing pronounced cytotoxicity to oxygen-deficient cells. This resulted in large differentials in the toxicities to hypoxic and aerobic cells (>1,000-fold at 10 μM). Low pH had only minimal effects on the cytotoxicity of KS119W. Under hypoxic conditions, EMT6 cells transfected to express high levels of either human or mouse versions of the repair protein O(6)-alkylguanine-DNA alkyltransferase, which is also known as O(6)-methylguanine DNA-methyltransferase, were much more resistant to KS119W than parental EMT6 cells lacking O(6)-alkylguanine-DNA alkyltransferase, confirming the importance of DNA O-6-alkylation to the cytotoxicity of this agent. Studies with EMT6 tumors in BALB/c Rw mice using both tumor cell survival and tumor growth delay assays showed that KS119W was effective as an adjunct to irradiation for the treatment of solid tumors in vivo, producing additive or supra-additive effects in most combination regimens for which the interactions could be evaluated. Our findings encourage additional preclinical studies to examine further the antineoplastic effects of KS119W alone and in combination with radiation, and to examine the pharmacology and toxicology of this new bioreductive alkylating agent so that its potential for clinical use as an adjuvant to radiotherapy can be evaluated.

Mapping DNA adducts of mitomycin C and decarbamoyl mitomycin C in cell lines using liquid chromatography/ electrospray tandem mass spectrometry.

The antitumor antibiotic and cancer chemotherapeutic agent mitomycin C (MC) alkylates and crosslinks DNA, forming six major MC-deoxyguanosine adducts of known structures in vitro and in vivo. Two of these adducts are derived from 2,7-diaminomitosene (2,7-DAM), a nontoxic reductive metabolite of MC formed in cells in situ. Several methods have been used for the analysis of MC-DNA adducts in the past; however, a need exists for a safer, more comprehensive and direct assay of the six-adduct complex. Development of an assay, based on mass spectrometry, is described. DNA from EMT6 mouse mammary tumor cells, Fanconi Anemia-A fibroblasts, normal human fibroblasts, and MCF-7 human breast cancer cells was isolated after MC or 10-decarbamoyl mitomycin C (DMC) treatment of the cells, digested to nucleosides, and submitted to liquid chromatography electrospray-tandem mass spectrometry. Two fragments of each parent ion were monitored ("multiple reaction monitoring"). Identification and quantitative analysis were based on a standard mixture of six adducts, the preparation of which is described here in detail. The lower limit of detection of adducts is estimated as 0.25 pmol. Three initial applications of this method are reported as follows: (i) differential kinetics of adduct repair in EMT6 cells, (ii) analysis of adducts in MC- or DMC-treated Fanconi Anemia cells, and (iii) comparison of the adducts generated by treatment of MCF-7 breast cancer cells with MC and DMC. Notable results are the following: Repair removal of the DNA interstrand cross-link and of the two adducts of 2,7-DAM is relatively slow; both MC and DMC generate DNA interstrand cross-links in human fibroblasts, Fanconi Anemia-A fibroblasts, and MCF-7 cells as well as EMT6 cells; and DMC shows a stereochemical preference of linkage to the guanine-2-amino group opposite from that of MC.

Synthesis and Photodynamic Activity of Novel Asymmetrically Substituted Fluorinated Phthalocyanines

A series of asymmetrically substituted dodecafluorinated phthalocyanines has been synthesized via the Kobayashi ring expansion reaction of the corresponding dodecafluorinated boron subphthalocyanine with differently substituted 1,3-diiminoisoindolines. The mild reaction conditions employed during this ring expansion reaction gave rise exclusively to 3:1 asymmetrically substituted dodecafluorinated phthalocyanines. Metal insertion into the metal-free phthalocyanines was accomplished by heating at 40 degrees C in N,N-dimethylformamide in the presence of zinc bromide. The resulting zinc dodecafluorophthalocyanines were formulated as Cremophor EL oil-water emulsions and evaluated as photosensitizers in vitro against EMT-6 mouse mammary tumor cells. As compared to the previously studied zinc hexadecafluorophthalocyanine, these new asymmetrical zinc dodecafluorophthalocyanines exhibited improved photodynamic activity.

131 Pharmacodynamic analysis of apoptosis and anti-vascular activity in GIST patients treated with Imatinib

To investigate the effects of texaphyrins on the oxygenation of EMT6 mouse mammary tumors in Balb/c Rw mice. Texaphyrins are synthetic, porphyrin-like molecules capable of stably coordinating lanthanide and nonlanthanide metals. Metallotexaphyrin compounds containing gadolinium (MGd), lutetium (MLu), europium (Eu-Tex), dysprosium (Dy-Tex), and manganese (Mn-Tex) were evaluated.Tumor oxygenation was measured using an Eppendorf pO2 histograph when tumors, implanted intradermally in the rear dorsum, reached 150–200 mm3. Oxygen measurements were also made in the leg muscle of tumor-bearing mice, to determine whether changes in oxygenation occurred in nontumor tissue.Motexafin gadolinium (Xcytrin, MGd) seems to be an effective modulator of tumor oxygen tension. The mean of the median tumor pO2 6 hours after injection of MGd was 8.0 ± 2.4 mm Hg. The control value was 1.5 ± 0.4 mm Hg. The oxygen levels within EMT6 tumors were shifted significantly toward higher oxygen tensions 6–8 hours after i.v. injection of 40 μmol/kg MGd, thereby reducing the percentage of severely hypoxic readings (MGd, 6 hours: 44.6 ± 4.3% <2.5 mm Hg; Control: 69.4 ± 3.0% <2.5 mm Hg). There was no significant change in the oxygenation of the leg muscle after MGd treatment. Eu-Tex and Mn-Tex increased the tumor oxygenation to a much lesser degree than MGd. MLu, Dy-Tex, and the vehicle (a 5% mannitol solution) did not modulate tumor oxygenation.MGd is an effective modulator of tumor oxygenation. The central metal composition of texaphyrin compounds is an important determinant of the effect of the texaphyrins on tumor oxygenation.

Effects of texaphyrins on the oxygenation of EMT6 mouse mammary tumors1, *1

<h2>Abstract</h2><h3>Purpose</h3> To investigate the effects of texaphyrins on the oxygenation of EMT6 mouse mammary tumors in Balb/c Rw mice. Texaphyrins are synthetic, porphyrin-like molecules capable of stably coordinating lanthanide and nonlanthanide metals. Metallotexaphyrin compounds containing gadolinium (MGd), lutetium (MLu), europium (Eu-Tex), dysprosium (Dy-Tex), and manganese (Mn-Tex) were evaluated. <h3>Methods</h3> Tumor oxygenation was measured using an Eppendorf pO₂ histograph when tumors, implanted intradermally in the rear dorsum, reached 150–200 mm³. Oxygen measurements were also made in the leg muscle of tumor-bearing mice, to determine whether changes in oxygenation occurred in nontumor tissue. <h3>Results</h3> Motexafin gadolinium (Xcytrin, MGd) seems to be an effective modulator of tumor oxygen tension. The mean of the median tumor pO₂ 6 hours after injection of MGd was 8.0 ± 2.4 mm Hg. The control value was 1.5 ± 0.4 mm Hg. The oxygen levels within EMT6 tumors were shifted significantly toward higher oxygen tensions 6–8 hours after i.v. injection of 40 μmol/kg MGd, thereby reducing the percentage of severely hypoxic readings (MGd, 6 hours: 44.6 ± 4.3% <2.5 mm Hg; Control: 69.4 ± 3.0% <2.5 mm Hg). There was no significant change in the oxygenation of the leg muscle after MGd treatment. Eu-Tex and Mn-Tex increased the tumor oxygenation to a much lesser degree than MGd. MLu, Dy-Tex, and the vehicle (a 5% mannitol solution) did not modulate tumor oxygenation. <h3>Conclusions</h3> MGd is an effective modulator of tumor oxygenation. The central metal composition of texaphyrin compounds is an important determinant of the effect of the texaphyrins on tumor oxygenation.

Effects of texaphyrins on the oxygenation of EMT6 mouse mammary tumors

Purpose To investigate the effects of texaphyrins on the oxygenation of EMT6 mouse mammary tumors in Balb/c Rw mice. Texaphyrins are synthetic, porphyrin-like molecules capable of stably coordinating lanthanide and nonlanthanide metals. Metallotexaphyrin compounds containing gadolinium (MGd), lutetium (MLu), europium (Eu-Tex), dysprosium (Dy-Tex), and manganese (Mn-Tex) were evaluated. Methods Tumor oxygenation was measured using an Eppendorf pO 2 histograph when tumors, implanted intradermally in the rear dorsum, reached 150–200 mm 3. Oxygen measurements were also made in the leg muscle of tumor-bearing mice, to determine whether changes in oxygenation occurred in nontumor tissue. Results Motexafin gadolinium (Xcytrin, MGd) seems to be an effective modulator of tumor oxygen tension. The mean of the median tumor pO 2 6 hours after injection of MGd was 8.0 ± 2.4 mm Hg. The control value was 1.5 ± 0.4 mm Hg. The oxygen levels within EMT6 tumors were shifted significantly toward higher oxygen tensions 6–8 hours after i.v. injection of 40 μmol/kg MGd, thereby reducing the percentage of severely hypoxic readings (MGd, 6 hours: 44.6 ± 4.3% <2.5 mm Hg; Control: 69.4 ± 3.0% <2.5 mm Hg). There was no significant change in the oxygenation of the leg muscle after MGd treatment. Eu-Tex and Mn-Tex increased the tumor oxygenation to a much lesser degree than MGd. MLu, Dy-Tex, and the vehicle (a 5% mannitol solution) did not modulate tumor oxygenation. Conclusions MGd is an effective modulator of tumor oxygenation. The central metal composition of texaphyrin compounds is an important determinant of the effect of the texaphyrins on tumor oxygenation.

Effects of RSR13 and oxygen on the cytotoxicity of cisplatin and carboplatin to EMT6 mouse mammary tumor cells in vitro and in vivo

Effects of RSR13 and oxygen on the cytotoxicity of cisplatin and carboplatin to EMT6 mouse mammary tumor cells in vitro and in vivo

Human SPF45, a Splicing Factor, Has Limited Expression in Normal Tissues, Is Overexpressed in Many Tumors, and Can Confer a Multidrug-Resistant Phenotype to Cells

Our effort to identify novel drug-resistant genes in cyclophosphamide-resistant EMT6 mouse mammary tumors led us to the identification of SPF45. Simultaneously, other groups identified SPF45 as a component of the spliceosome that is involved in alternative splicing. We isolated the human homologue and examined the normal human tissue expression, tumor expression, and the phenotype caused by overexpression of human SPF45. Our analyses revealed that SPF45 is expressed in many, but not all, normal tissues tested with predominant expression in normal ductal epithelial cells of the breast, liver, pancreas, and prostate. Our analyses using tissue microarrays and sausages of tumors indicated that SPF45 is highly expressed in numerous carcinomas including bladder, breast, colon, lung, ovarian, pancreatic, and prostate. Interestingly, this study revealed that overexpression of SPF45 in HeLa, a cervical carcinoma cell line, resulted in drug resistance to doxorubicin and vincristine, two chemotherapeutic drugs commonly used in cancer. To our knowledge, this is the first study showing tumor overexpression of an alternate splicing factor resulting in drug resistance.

Effects of RSR13 and oxygen on the cytotoxicity of cisplatin and carboplatin to EMT6 mouse mammary tumor cells in vitro and in vivo.

RSR13, 2-[4-[2-[(3,5-dimethylphenyl)amino]-2-oxoethyl]phenoxy]-2-methylpropanoic acid monosodium salt, allosterically modifies hemoglobin to increase tumor pO(2), increases the effect of radiation in animal tumor models, and is in phase III clinical trials as an adjuvant to radiotherapy. Cisplatin and carboplatin, two commonly used anticancer drugs have been used in combination with radiotherapy. Some studies have suggested that the cytotoxic effects of these drugs are altered in hypoxia. This study tested whether RSR13 plus oxygen breathing increased the cytotoxicity of cisplatin and carboplatin in vivo. Solid EMT6 tumors in BALB/c Rw mice were treated with cisplatin (5-30 microg/g) or carboplatin (5-200 microg/g) along with 150 microg/g RSR13 in combination with oxygen breathing. Tumor cell survival was assayed using clonogenic assays. The effects of pre- and posttreatments with RSR13 and oxygen breathing on the cytotoxicity of cisplatin or carboplatin were also examined. To assess whether RSR13 had direct effects on the cytotoxicity of the drugs, exponentially growing monolayers of EMT6 mouse mammary carcinoma cells were treated with graded concentrations of cisplatin or carboplatin for 2 h along with simultaneous (2 h) RSR13 treatments or with prolonged (22 h) pre- or posttreatment incubations with 100 microM RSR13. Single or multiple treatments with 150 microg/g RSR13 plus oxygen breathing had no effect on the viability of cells in EMT6 tumors in mice. After treatment with cisplatin or carboplatin, the tumor cell survival tended to be lower in oxygen-breathing mice especially at higher doses of cisplatin. Treatment with RSR13 plus oxygen breathing beginning 15 min before administration of the alkylating agents did not alter the cytotoxicity of cisplatin or carboplatin from that seen with oxygen breathing alone. Pretreatment with RSR13 plus oxygen at 22 and 14 h prior to administration of either cisplatin or carboplatin did not alter the effect of either alkylating agent. Treatment with RSR13 plus oxygen breathing beginning 15 min before administration of the alkylating agents and lasting for 2 or 5 h did not alter the cytotoxicity of either drug from that seen with oxygen breathing alone. The cytotoxicity of cisplatin was not altered by treatment with oxygen alone or with RSR13 plus oxygen for 5 h after cisplatin injection. For carboplatin, treatment with oxygen alone and with RSR13 plus oxygen for 5 h after injection increased to similar extents the response of the tumor cells compared to that seen with assays at 2 h. Neither short simultaneous treatments, prolonged pretreatment incubations, nor prolonged posttreatment incubations with RSR13 altered the survival of EMT6 cells in cultures treated with cisplatin or carboplatin. These findings indicate that RSR13 in combination with oxygen breathing does not alter the cytotoxicity of cisplatin or carboplatin when used simultaneously, as a pretreatment or as a posttreatment in vitro or in vivo. Our in vivo findings indicate trends that support previous findings that cisplatin is more cytotoxic to well-oxygenated tumor cells than to hypoxic tumor cells, and that this effect can be improved by improving tumor oxygenation, but the differences seen in our studies did not achieve statistical significance.

Preliminary studies of the effects of gadolinium texaphyrin on the growth and radiosensitivity of EMT6 cells in vitro

Purpose: To investigate the effects of gadolinium texaphyrin (GdTx) on the growth and radiation response of cells in vitro, in a limited set of experiments designed to examine some areas of controversy concerning the effects of this compound.Methods and Materials: Exponentially growing cultures of EMT6 mouse mammary tumor cells, grown in Dulbecco’s Modified Eagle’s Medium with 10% dialyzed fetal bovine serum, were treated with GdTx either prepared from powder or obtained as a solution similar to that used clinically, in either the presence or absence of equimolar ascorbic acid. Cell viability was measured using a clonogenic assay.Results: Treatment with GdTx in the presence of ascorbic acid dramatically altered the growth, appearance, and behavior of the cells; treatment with GdTx in the absence of ascorbic acid had only minimal effects. The effects of the powdered drug and the solution were similar. GdTx used with equimolar ascorbic acid altered the radiation dose-response curves of cells irradiated under aerobic and hypoxic conditions; no significant changes were observed without ascorbic acid.Conclusions: The details of the protocols used in experiments examining the effects of GdTx have major effects on the outcomes. Our results suggest that differences in the protocols used by different groups in past studies with GdTx probably were important in producing the disparate results reported previously.

Photodynamic properties of amphiphilic derivatives of aluminum tetrasulfophthalocyanine.

Photodynamic therapy (PDT) is a promising treatment modality that has recently been accepted in clinics as a curative or palliative therapy for cancer and other nonmalignant conditions. Phthalocyanines (Pc) are attractive photosensitizers for PDT because of their enhanced photophysical and photochemical properties. The overall charge and solubility of Pc play a major role in their potential usefulness for PDT. A series of amphiphilic derivatives of tetrasulfonated aluminum Pc (AlPcS4) was prepared by substituting one of the four sulfonate groups with aliphatic side chains of 4, 8, 12 and 16 carbon atoms. The photodynamic properties of the derivatives were compared with those of AlPcS4 and the adjacent disulfonated aluminum Pc. Parameters studied included reversed-phase high-performance liquid chromatography (HPLC) retention times, capacity to generate singlet oxygen (1O2), in vitro cell uptake and phototoxicity, as well as PDT response of transplantable EMT-6 tumors in mice. The monomerized AlPcS4 derivatives showed similar or higher capacities to generate 1O2 as compared with the parent AlPcS4 as measured from relative L-tryptophan photooxidation yields. A549 cell uptake of the AlPcS4 derivatives decreased in the following order: AlPcS4(C16) > AlPcS4(C12) > AlPcS4(C8) > AlPcS4(C4). Human low-density lipoprotein at high concentrations (40 micrograms/mL) completely prevented uptake, whereas at 4 micrograms/mL uptake was decreased for the more lipophilic compounds and yet remained unaffected for the more hydrophilic dyes. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, A549 cell survival was assessed; it showed that photocytotoxic activity varied directly with the HPLC retention times, i.e. more hydrophilic compounds were less phototoxic. As 1O2 yields were similar for the four substituted AlPcS4 derivatives, it was postulated that the increased cytotoxic activity was caused by enhanced subcellular localization as a result of the long aliphatic side chains. These amphiphilic compounds proved to be photodynamically potent against the EMT-6 mouse mammary tumor model implanted in Balb/c mice. At dye doses of 0.2 mumol/kg and a fluence of 400 J/cm2 complete tumor regression was observed with no morbidity. The substitution of AlPcS4 with long aliphatic chains on the macrocycle greatly enhances its photodynamic efficacy both in vitro and in vivo.

Optimizing pH-responsive Polymeric Micelles for Drug Delivery in a Cancer Photodynamic Therapy Model

Different pH-sensitive, randomly- and terminally-alkylated N -isopropylacrylamide (NIPAM) copolymers were synthesized and used to prepare pH-responsive polymeric micelles (PM). These copolymers were modified from previously-studied copolymers by incorporating an additional hydrophilic monomer, N- vinyl-2-pyrrolidone (VP) to decrease uptake by the mononuclear phagocyte system (MPS) and improve localization in tumors. VP lowered the phase transition pH of the copolymers but did not affect the onset of micellization. The in vitro cytotoxicity of the copolymers was evaluated on EMT-6 mouse mammary tumor cells in comparison to Cremophor EL (CRM). The anticancer photosensitizer aluminum chloride phthalocyanine (AlClPc) was loaded into the PM with a standard dialysis procedure. Biodistribution and in vivo photodynamic activity were then evaluated in Balb/c mice bearing intradermal EMT-6 tumors. All NIPAM copolymers demonstrated substantially lower cell cytotoxicity than the control surfactant CRM. In vivo, similar AlClPc tumor uptake was observed for the PM and CRM formulations. However, the PM appeared to exhibit greater activity in vivo than CRM formulation at an AlClPc subtherapeutic dose. Therefore, NIPAM-based copolymers containing VP units represent promising alternatives for the formulation of poorly water-soluble phthalocyanines.

Prostaglandin A1 inhibits stress-induced NF-kappaB activation and reverses resistance to topoisomerase II inhibitors.

Stress conditions associated with solid tumors lead to the formation of heterogeneous tumor cell subpopulations and insensitivity to cancer chemotherapeutics. In this report, we show that EMT6 mouse mammary tumor cells treated with the chemical stress, brefeldin A (BFA), or the physiological stress, hypoxia, develop resistance to the topoisomerase II (topoII) inhibitors teniposide and etoposide. BFA and hypoxia treatment did not alter intracellular drug concentrations, topoll protein levels, or inhibit topoII activity. BFA and hypoxia did cause the activation of the nuclear transcription factor NF-kappaB. We demonstrate that pretreatment with the synthetic cyclopentenone prostaglandin A1 (PGA1) inhibits stress-induced NF-kappaB activation and reverses BFA- and hypoxia-induced resistance. The reversal of BFA-induced resistance can occur when PGA1 is administered either before or several hours after the induction of stress. Taken together, these data support the involvement of NF-kappaB in stress-induced drug resistance, show that pharmacologic inhibitors of NF-kappaB can disrupt the biological consequences of stress, and imply that inhibitors of NF-kappaB may be useful agents to enhance the clinical efficacy of topoII-directed chemotherapeutics.

Bioreductive metabolism of mitomycin C in EMT6 mouse mammary tumor cells: cytotoxic and non-cytotoxic pathways, leading to different types of DNA adducts. The effect of dicumarol

The six DNA adducts formed in EMT6 mouse mammary tumor cells upon treatment with mitomycin C (MC) fall into two groups: (1) four guanine adducts of MC and (2) two guanine adducts derived from 2,7-diaminomitosene (2,7-DAM), the major reductive metabolite of MC. The two groups of adducts were proposed to originate from two pathways arising from reductive activation of MC: (a) direct alkylation of DNA and (b) formation of 2,7-DAM, which then alkylates DNA. The aim of this study was to test the validity of this proposal and to evaluate the significance of alkylation of DNA by 2,7-DAM. Treatment of the cells with 2,7-DAM itself yielded the same 2,7-DAM-guanine adducts as treatment with MC; however, 2,7-DAM was approximately 100-fold less cytotoxic than MC. The uptake and efflux of 2,7-DAM by EMT6 cells was comparable to that of MC, but 2,7-DAM alkylated DNA with higher efficiency than MC. These results validate the two proposed pathways and show that formation of 2,7-DAM-DNA adducts in MC-treated cells represents a relatively non-toxic pathway of reductive metabolism of MC. A selective stimulatory effect of dicumarol (DIC) on 2,7-DAM-DNA adduct formation in EMT6 cells treated with MC was also investigated. DIC had no effect on alkylation by MC in cell-free systems, nor did it have significant effects on adduct formation or cell survival for cells treated with 2,7-DAM. It is proposed that in the cell DIC stimulates a reductase enzyme located at subcellular sites where the activated MC species has no direct access to DNA and therefore is diverted into the non-cytotoxic pathway, which leads to the formation of 2,7-DAM and its adducts.

In-vitro and in-vivo evaluation of pH-responsive polymeric micelles in a photodynamic cancer therapy model.

pH-sensitive polymeric micelles of randomly and terminally alkylated N-isopropylacrylamide copolymers were prepared and characterized. Aluminium chloride phthalocyanine (AlClPc), a second generation sensitizer for the photodynamic therapy of cancer, was incorporated in the micelles by dialysis. Their photodynamic activities were evaluated in-vitro against EMT-6 mouse mammary tumour cells and in-vivo against EMT-6 tumours implanted intradermally on each hind thigh of Balb/c mice. pH-sensitive polymeric micelles were found to exhibit greater cytotoxicity in-vitro than control Cremophor EL formulations. In the presence of chloroquine, a weak base that raises the internal pH of acidic organelles, in-vitro experiments demonstrated the importance of endosomalllysosomal acidity for the pH-sensitive polymeric micelles to be fully effective. Biodistribution was assessed by fluorescence of tissue extracts after intravenous injection of 2 micromol kg(-1) AlClPc. The results revealed accumulation of AlClPc polymeric micelles in the liver, spleen and lungs, with a lower tumour uptake than AlClPc Cremophor EL formulations. However, polymeric micelles exhibited similar activity in-vivo to the control Cremophor EL formulations, demonstrating the higher potency of AlClPc polymeric micelles when localized in tumour tissue. It was concluded that polymeric micelles represent a good alternative to Cremophor EL preparations for the vectorization of hydrophobic drugs.

Low-density lipoprotein-bound aluminum sulfophthalocyanine: targeting tumor cells for photodynamic therapy

The aim of this study was to evaluate the role of low-density lipoproteins (LDLs) as a vehicle for aluminum sulfophthalocyanine ( AlPcS ) to target tumor cells for photodynamic therapy (PDT). LDLs are biological particles containing an apolipoprotein which recognizes with high affinity specific receptors on many cell types, including cancer cells. LDL was loaded with AlPcS in two different manners. In the first procedure, the tetrasulfonated AlPcS4was covalently bound to the protein part of the LDL via amide bonds to 6-carboxypentylaminosulfonyl spacer chains attached to two of the four sulfonate groups, i.e. AlPcS4A2. In the second procedure, the AlPcS4was substituted with a linear dodecylaminosulfonyl chain, i.e. AlPcS4( C12) and non-covalently inserted into the phospholipid monolayer of the LDL. Both preparations contained over 50 moles AlPcS /mole LDL. They were tested in vitro for their phototoxic properties against the EMT-6 mouse mammary tumor cell line and the A-549 human lung adenocarcinoma cell line. Cell survival was assessed using the MTT colorimetric assay. Association of the free AlPcS4( C12) with LDL increased the in vitro phototoxicity of the dye substantially against both cell lines while the covalently loaded AlPcS4A2-LDL preparation showed little cytotoxicity, even at a 10-fold-higher light or drug doses. It was postulated that the covalent labeling of the protein moiety with the Pc greatly reduced LDL receptor recognition, rendering this derivative photo-inactive. Photodynamic therapy of EMT-6 tumor-bearing mice showed that the free and LDL-associated AlPcS4( C12) exhibited similar activities, with 100% cure one week post-PDT at 0.2 μmol kg−1. We conclude that the attached aliphatic chain of the AlPcS4( C12) greatly enhances the phototoxicity of the parent AlPcS4but that pre-association of the AlPcS4( C12) with LDL does not further augment its in vivo photodynamic efficacy.

Structure of adduct X, the last unknown of the six major DNA adducts of mitomycin C formed in EMT6 mouse mammary tumor cells.

Treatment of EMT6 mouse mammary tumor cells with mitomycin C (MC) results in the formation of six major MC-DNA adducts. We identified the last unknown of these ("adduct X") as a guanine N(2) adduct of 2, 7-diaminomitosene (2,7-DAM), in which the mitosene is linked at its C-10 position to guanine N(2). The assigned structure is based on UV and mass spectra of adduct X isolated directly from the cells, as well as on its difference UV, second-derivative UV, and circular dichroism spectra, synthesis from [8-(3)H]deoxyguanosine, and observation of its heat stability. These tests were carried out using 17 microg of synthetic material altogether. The mechanism of formation of adduct X involves reductive metabolism of MC to 2,7-DAM, which undergoes a second round of reductive activation to alkylate DNA, yielding adduct X and another 2,7-DAM-guanine adduct (adduct Y), which is linked at guanine N7 to the mitosene. Adduct Y has been described previously. Adduct X is formed preferentially at GpC, while adduct Y favors the GpG sequence. In contrast to MC-DNA adducts, the 2,7-DAM-DNA adducts are not cytotoxic.

Water-soluble aluminium phthalocyanine-polymer conjugates for PDT: photodynamic activities and pharmacokinetics in tumour-bearing mice.

The potential use of unsubstituted aluminium phthalocyanine (AlClPc) as a sensitizer for photodynamic therapy (PDT) of cancer has not been fully exploited in spite of its higher efficiency as compared to the sulphonated derivatives. This is largely due to the strong hydrophobic character of AlClPc which renders the material difficult to formulate for in vivo administration. We prepared two water-soluble derivatives of AlClPc by axial coordination of polyethyleneglycol (PEG, MW 2000) or polyvinylalcohol (PVA, MW 13 000–23 000) to the central aluminium ion. Their photodynamic activities were evaluated in vitro against the EMT-6 mouse mammary tumour cells and in vivo against the EMT-6 and the colon carcinoma Colo-26 tumours implanted intradermally in Balb/c mice. Pharmacokinetics were studied in the EMT-6 tumour-bearing mice. After 1 h incubation, the light dose required to kill 90% of cells (LD90) was at least three times less for AlClPc (Cremophor emulsion) as compared to AlPc–PEG and AlPc–PVA, while after 24 h incubation all three preparations were highly phototoxic. All three dye preparations induced complete EMT-6 tumour regression in 75–100% of animals at a low drug dose (0.25 μmol kg−1) following PDT (400 J cm−2, 650–700 nm) at 24 h pi. Complete tumour regression in the Colo-26 tumour model was obtained in 30% of mice at a dose of 2 μmol kg−1. In the non-cured animals, AlPc–PVA induced the most significant tumour growth delay. This dye showed a prolonged plasma half-life (6.8 h) as compared to AlClPc (2.6 h) and AlPc–PEG (23 min), lower retention by liver and spleen and higher tumour-to-skin and tumour-to-muscle ratios. Our data demonstrate that addition of hydrophilic axial ligands to AlPc, while modifying in vitro and in vivo kinetics, does not reduce the PDT efficiency of the parent molecule. Moreover, in the case of the polyvinylalcohol derivative, axial coordination confers advantageous pharmacokinetics to AlPc, which makes this photosensitizer a valuable, water soluble candidate drug for clinical PDT of cancer. © 1999 Cancer Research Campaign