EMT6 Mouse Mammary Carcinoma Research Articles

Abstract 6200: PARP1 hyperactivation by the decoy oligodeoxynucleotide OX425 mediates DNA repair abrogation and unleashes the anti-tumor immune response

Abstract Background: Poly (ADP-ribose) polymerase inhibitors (PARPis) lead to synthetic lethality when used in cancers with homologous recombination deficiency (HRD). However, the development of resistance to PARPis is a recurrent problem thus limits the duration of response and hence the clinical utility of these agents. Here, we describe the antineoplastic and immunomodulatory effects of OX425, a first-in-class oligodeoxynucleotide that operates as a PARP1 decoy, resulting in constitutive PARP1 hyperactivation and consequent exhaustion of the DNA damage response. Methods: OX425-induced PARP trapping, hyperactivation and cell cytotoxicity were examined in vitro in HRD and homologous recombination proficient (HRP) human cancer cells, as well as in non-transformed cell lines. DNA repair efficacy was monitored by analyzing repair protein recruitment to damage sites. OX425 effects on the innate and adaptive immune responses were assessed by following STING activation and T-cell mediated anti-tumor cytotoxicity. RNAseq analysis in HRP/HRD tumor cells treated with OX425 or PARP inhibitors was employed to uncover the molecular mechanisms underlying OX425 effects. The anticancer efficacy of OX425 was assessed in vivo in different HRD and HRP tumor models. OX425-induced PARP activation and tumor infiltration by immune cells were analyzed by flow cytometry. Results: At odds with conventional PARP inhibitors, OX425 bound to and hyperactivated PARP1 with high affinity in a dose-dependent manner, resulting in elevated cytotoxicity to multiple cancer cells (breast, ovarian, prostate, colon, hematological, endometrial cancers) irrespective of HR status. Interestingly, long-term treatment with OX425 did not show any mutagenicity compared to PARPi. The activity of OX425 was specific to tumor cells, as no significant effect on cell viability was observed for normal cells, at odds with PARP inhibitors. In line with in vitro results, OX425 mediated considerable anticancer effects in vivo. Moreover, OX425 triggered activation of the STING pathway and CCL5 secretion in the EMT6 mouse mammary carcinoma model. The anticancer effect of OX425 was coupled with tumor-targeting T cell responses. In MPA/DMBA-driven mammary tumors, OX425 mediated considerable anticancer effects in monotherapy and synergistic effects in combination with PD1 inhibition. Moreover, OX425 treatment significantly delayed acquired resistance to olaparib in BRCA1 mutated MDA-MB-436 cell-derived xenografts. Conclusions: Our results provide preclinical rationale for using OX425 to trigger DNA damage exhaustion and STING activation in cancer cells and initiate inflammatory responses that can be actioned by immune checkpoint inhibitors in patients bearing HRD or HRP tumors Citation Format: Vlada Zakharova, Claudia Galassi, Chloé Doizelet, Vincent Hayes, Lorenzo Galluzzi, Wael Jdey. PARP1 hyperactivation by the decoy oligodeoxynucleotide OX425 mediates DNA repair abrogation and unleashes the anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6200.

Acquired radioresistance in EMT6 mouse mammary carcinoma cell line is mediated by CTLA-4 and PD-1 through JAK/STAT/PI3K pathway

Cancer recurrence is often associated with the acquisition of radioresistance by cancer tissues due to failure in radiotherapy. The underlying mechanism leading to the development of acquired radioresistance in the EMT6 mouse mammary carcinoma cell line and the potential pathway involved was investigated by comparing differential gene expressions between parental and acquired radioresistance cells. EMT6 cell line was exposed to 2 Gy/per cycle of gamma-ray and the survival fraction between EMT6-treated and parental cells was compared. EMT6RR_MJI (acquired radioresistance) cells was developed after 8 cycles of fractionated irradiation. The development of EMT6RR_MJI cells was confirmed with further irradiation at different doses of gamma-ray, and both the survival fraction and migration rates were measured. Higher survival fraction and migration rates were obtained in EMT6RR_MJI cells after exposure to 4 Gy and 8 Gy gamma-ray irradiations compared to their parental cells. Gene expression between EMT6RR_MJI and parental cells was compared, and 16 genes identified to possess more than tenfold changes were selected and validated using RT-PCR. Out of these genes, 5 were significantly up-regulated i.e., IL-6, PDL-1, AXL, GAS6 and APCDD1. Based on pathway analysis software, the development of acquired radioresistance in EMT6RR_MJI was hypothesized through JAK/STAT/PI3K pathway. Presently, CTLA-4 and PD-1 were determined to be associated with JAK/STAT/PI3K pathway, where both their expressions were significantly increased in EMT6RR_MJI compared to parental cells in the 1st, 4th and 8th cycle of radiation. As a conclusion, the current findings provided a mechanistic platform for the development of acquired radioresistance in EMT6RR_MJI through overexpression of CTLA-4 and PD-1, and novel knowledge on therapeutic targets for recurrent radioresistant cancers.

Tumor cell lysate-loaded immunostimulatory spherical nucleic acids as therapeutics for triple-negative breast cancer

Highly heterogenous cancers, such as triple-negative breast cancer (TNBC), remain challenging immunotherapeutic targets. Herein, we describe the synthesis and evaluation of immunotherapeutic liposomal spherical nucleic acids (SNAs) for TNBC therapy. The SNAs comprise immunostimulatory oligonucleotides (CpG-1826) as adjuvants and encapsulate lysates derived from TNBC cell lines as antigens. The resulting nanostructures (Lys-SNAs) enhance the codelivery of adjuvant and antigen to immune cells when compared to simple mixtures of lysates with linear oligonucleotides both in vitro and in vivo, and reduce tumor growth relative to simple mixtures of lysate and CpG-1826 (Lys-Mix) in both Py230 and Py8119 orthotopic syngeneic mouse models of TNBC. Furthermore, oxidizing TNBC cells prior to lysis and incorporation into SNAs (OxLys-SNAs) significantly increases the activation of dendritic cells relative to their nonoxidized counterparts. When administered peritumorally in vivo in the EMT6 mouse mammary carcinoma model, OxLys-SNAs significantly increase the population of cytotoxic CD8+ T cells and simultaneously decrease the population of myeloid derived suppressor cells (MDSCs) within the tumor microenvironment, when compared with Lys-SNAs and simple mixtures of oxidized lysates with CpG-1826. Importantly, animals administered OxLys-SNAs exhibit significant antitumor activity and prolonged survival relative to all other treatment groups, and resist tumor rechallenge. Together, these results show that the way lysates are processed and packaged has a profound impact on their immunogenicity and therapeutic efficacy. Moreover, this work points toward the potential of oxidized tumor cell lysate-loaded SNAs as a potent class of immunotherapeutics for cancers lacking common therapeutic targets.

PH-Sensitive PEGylated liposomes for delivery of an acidic dinitrobenzamide mustard prodrug: Pathways of internalization, cellular trafficking and cytotoxicity to cancer cells

This paper aims to develop and evaluate a pH-sensitive PEGylated liposomal (pPSL) system for tumor-targeted intracellular delivery of SN25860, a weakly acidic, poorly water-soluble dinitrobenzamide mustard prodrug which is activated by the E. coli nitroreductase nfB. pPSL and non pH-sensitive liposomes (nPSL), as reference, were formulated by thin-film hydration; an active drug loading method was developed with the aid of solubilizers. Cytotoxicity was evaluated in an nfsB-transfected EMT6 mouse mammary carcinoma cell line. Cellular uptake of liposomes was evaluated by both high performance liquid chromatography and flow cytometry. Intracellular trafficking was visualised by confocal microscopy. High drug loading (7.0±0.2% w/w) was achieved after systematic optimization of drug loading conditions. pPSL-SN25860 demonstrated a 21 and 24- fold increase in antiproliferative potency compared to nPSL-SN25860 and free drug, respectively. Cells treated with pPSL had a 1.6–2.5- fold increase in intracellular drug concentration compared to nPSL. This trend was consistent with flow cytometry results. Cells treated with chlorpromazine demonstrated reduced uptake of both nPSL (40%) and pPSL (46%), indicating clathrin-mediated endocytosis was the major pathway. Confocal microscopy showed that pPSL had not only undergone faster and greater endocytosis than nPSL but was also homogeneously distributed in the cytosol and nuclei suggesting endosome escape, in contrast to nPSL.

Chloroethylating and methylating dual function antineoplastic agents display superior cytotoxicity against repair proficient tumor cells

Two new agents based upon the structure of the clinically active prodrug laromustine were synthesized. These agents, 2-(2-chloroethyl)-N-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (1) and N-(2-chloroethyl)-2-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (2), were designed to retain the potent chloroethylating and DNA cross-linking functions of laromustine, and gain the ability to methylate DNA at the O-6 position of guanine, while lacking the carbamoylating activity of laromustine. The methylating arm was introduced with the intent of depleting the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT). Compound 1 is markedly more cytotoxic than laromustine in both AGT minus EMT6 mouse mammary carcinoma cells and high AGT expressing DU145 human prostate carcinoma cells. DNA cross-linking studies indicated that its cross-linking efficiency is nearly identical to its predicted active decomposition product, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which is also produced by laromustine. AGT ablation studies in DU145 cells demonstrated that 1 can efficiently deplete AGT. Studies assaying methanol and 2-chloroethanol production as a consequence of the methylation and chloroethylation of water by 1 and 2 confirmed their ability to function as methylating and chloroethylating agents and provided insights into the superior activity of 1.

KS900: A hypoxia-directed, reductively activated methylating antitumor prodrug that selectively ablates O 6-alkylguanine-DNA alkyltransferase in neoplastic cells

To most effectively treat cancer it may be necessary to preferentially destroy tumor tissue while sparing normal tissues. One strategy to accomplish this is to selectively cripple the involved tumor resistance mechanisms, thereby allowing the affected anticancer drugs to gain therapeutic efficacy. Such an approach is exemplified by our design and synthesis of the intracellular hypoxic cell activated methylating agent, 1,2-bis(methylsulfonyl)-1-methyl-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS900) that targets the O-6 position of guanine in DNA. KS900 is markedly more cytotoxic in clonogenic experiments under conditions of oxygen deficiency than the non-intracellularly activated agents KS90, and 90M, when tested in O 6-alkylguanine-DNA alkyltransferase (AGT) non-expressing cells (EMT6 mouse mammary carcinoma, CHO/AA8 hamster ovary, and U251 human glioma), and than temozolomide when tested in AGT expressing cells (DU145 human prostate carcinoma). Furthermore, KS900 more efficiently ablates AGT in HL-60 human leukemia and DU145 cells than the spontaneous globally activated methylating agent KS90, with an IC 50 value over 9-fold lower than KS90. Finally, KS900 under oxygen-deficient conditions selectively sensitizes DU145 cells to the chloroethylating agent, onrigin, through the ablation of the resistance protein AGT. Thus, under hypoxia, KS900 is more cytotoxic at substantially lower concentrations than methylating agents such as temozolomide that are not preferentially activated in neoplastic cells by intracellular reductase catalysts. The necessity for intracellular activation of KS900 permits substantially greater cytotoxic activity against cells containing the resistance protein O 6-alkylguanine-DNA alkyltransferase (AGT) than agents such as temozolomide. Furthermore, the hypoxia-directed intracellular activation of KS900 allows it to preferentially ablate AGT pools under the oxygen-deficient conditions that are present in malignant tissue.

Down-regulation of DNA mismatch repair proteins in human and murine tumor spheroids: implications for multicellular resistance to alkylating agents

Similar to other anticancer agents, intrinsic or acquired resistance to DNA-damaging chemotherapeutics is a major obstacle for cancer therapy. Current strategies aimed at overcoming this problem are mostly based on the premise that tumor cells acquire heritable genetic mutations that contribute to drug resistance. Here, we present evidence for an epigenetic, tumor cell adhesion-mediated, and reversible form of drug resistance that is associated with a reduction of DNA mismatch repair proteins PMS2 and/or MLH1 as well as other members of this DNA repair process. Growth of human breast cancer, human melanoma, and murine EMT-6 breast cancer cell lines as multicellular spheroids in vitro, which is associated with increased resistance to many chemotherapeutic drugs, including alkylating agents, is shown to lead to a reproducible down-regulation of PMS2, MLH1, or, in some cases, both as well as MHS6, MSH3, and MSH2. The observed down-regulation is in part reversible by treatment of tumor spheroids with the DNA-demethylating agent, 5-azacytidine. Thus, treatment of EMT-6 mouse mammary carcinoma spheroids with 5-azacytidine resulted in reduced and/or disrupted cell-cell adhesion, which in turn sensitized tumor spheroids to cisplatin-mediated killing in vitro. Our results suggest that antiadhesive agents might sensitize tumor spheroids to alkylating agents in part by reversing or preventing reduced DNA mismatch repair activity and that the chemosensitization properties of 5-azacytidine may conceivably reflect its role as a potential antiadhesive agent as well as reversal agent for MLH1 gene silencing in human tumors.

Registration of [ 18F]FDG microPET and small-animal MRI

This paper describes a voxel-based method for coregistering microPET [ 18F]FDG emission images and MRI data without the need for fiducial markers. [ 18F]FDG has a well-characterized biodistribution in normal mice and thus may be useful for image registration. Female BALB/c mice were implanted with EMT-6 mouse mammary carcinoma 1 week prior to imaging. Three imaging sessions were performed in which a [ 18F]FDG microPET-R4 emission scan was taken followed by small-animal MRI with and without Gd-based contrast agent. MicroPET and MR images were registered using a voxel-based algorithm that computes rigid-body image transformations based on the alignment of intensity gradients. Registration accuracy was assessed on the basis of dual-modality external fiducial line sources incorporated into the mouse bed. The root mean square (rms) registration errors were 0.74 mm translation and 1.44° rotation without contrast and 0.72 mm translation and 0.89° rotation with contrast. Generally, good registration was evident upon inspection of fused microPET/MR images. Accurate automated, voxel-based microPET–MR image coregistration, utilizing image intensity gradients, is feasible. Our technique requires no manual identification of image features and makes no use of surgically implanted or external fiducial markers or stereotactic apparatus.

Effects of motexafin gadolinium on DNA damage and X-ray-induced DNA damage repair, as assessed by the Comet assay

To investigate the effects of motexafin gadolinium (MGd) on the levels of reactive oxygen species (ROS), glutathione (GSH), and DNA damage in EMT6 mouse mammary carcinoma cells. The ability of MGd to alter radiosensitivity and to inhibit DNA damage repair after X-ray irradiation was also evaluated. Reactive oxygen species and GSH levels were assessed by 2,7-dichlorofluorescein fluorescence flow cytometry and the Tietze method, respectively. Cellular radiosensitivity was assessed by clonogenic assays. Deoxyribonucleic acid damage and DNA damage repair were assessed in plateau-phase EMT6 cells by the Comet assay and clonogenic assays. Cells treated with 100 mumol/L MGd plus equimolar ascorbic acid (AA) had significantly increased levels of ROS and a 58.9% +/- 3.4% decrease in GSH levels, relative to controls. Motexafin gadolinium plus AA treatment increased the hypoxic, but not the aerobic, radiosensitivity of EMT6 cells. There were increased levels of single-strand breaks in cells treated with 100 mumol/L MGd plus equimolar AA, as evidenced by changes in the alkaline tail moment (MGd + AA, 6 h: 14.7 +/- 1.8; control: 2.8 +/- 0.9). The level of single-strand breaks was dependent on the length of treatment. Motexafin gadolinium plus AA did not increase double-strand breaks. The repair of single-strand breaks at 2 h, but not at 4 h and 6 h, after irradiation was altered significantly in cells treated with MGd plus AA (MGd + AA, 2 h: 15.8 +/- 3.4; control: 5.8 +/- 0.6). Motexafin gadolinium did not alter the repair of double-strand breaks at any time after irradiation with 10 Gy. Motexafin gadolinium plus AA generated ROS, which in turn altered GSH homeostasis and induced DNA strand breaks. The MGd plus AA-mediated alteration of GSH levels increased the hypoxic, but not aerobic, radiosensitivity of EMT6 cells. Motexafin gadolinium altered the kinetics of single-strand break repair soon after irradiation but did not inhibit potentially lethal damage repair in EMT6 cells.

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine: An anticancer agent targeting hypoxic cells

To target malignant cells residing in hypoxic regions of solid tumors, we have designed and synthesized prodrugs generating the cytotoxic alkylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) after bioreductive activation. We postulate that one of these agents, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119), requires enzymatic nitro reduction to produce 90CE, whereas another agent, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(4-nitrobenzyloxy)carbonyl]hydrazine (PNBC), can also be activated by nucleophilic attack by thiols such as glutathione (GSH)/GST. We demonstrated that these agents selectively kill hypoxic EMT6 mouse mammary carcinoma and CHO cells. In hypoxia, 50 microM KS119 produced 5 logs of kill of EMT6 cells without discernable cytotoxicity in air; similar effects were observed with CHO cells. PNBC was less efficacious against hypoxic tumor cells and also had some toxicity to aerobic cells, presumably because of GST/thiol activation, making PNBC less interesting as a selective hypoxic-cell cytotoxin. BALB/c mice with established EMT6 solid tumors were used to demonstrate that KS119 could reach and kill hypoxic cells in solid tumors. To gain information on bioreductive enzymes involved in the activation of KS119, cytotoxicity was measured in CHO cell lines overexpressing NADH:cytochrome b5 reductase (NBR), NADPH:cytochrome P450 reductase (NPR), or NADPH: quinone oxidoreductase 1 (NQO1). Increased cytotoxicity occurred in cells overexpressing NBR and NPR, whereas overexpressed NQO1 had no effect. These findings were supported by enzymatic studies using purified NPR and xanthine oxidase to activate KS119. KS119 has significant potential as a hypoxia-selective tumor-cell cytotoxin and is unlikely to cause major toxicity to well oxygenated normal tissues.

Investigation into 64Cu-labeled Bis(selenosemicarbazone) and Bis(thiosemicarbazone) complexes as hypoxia imaging agents

Background Cu-diacetyl-bis( N 4-methylthiosemicarbazone) [Cu-ATSM], although excellent for oncology applications, may not be suitable for delineating cardiovascular or neurological hypoxia. For this reason, new Cu hypoxia positron emission tomography (PET) imaging agents are being examined to search for a higher selectivity for hypoxic or ischemic tissue at higher oxygen concentrations found in these tissues. Two approaches are to increase alkylation or to replace the sulfur atoms with selenium, resulting in the formation of selenosemicarbazones. Methods Three 64Cu-labeled selenosemicarbazone complexes were synthesized and one was screened for hypoxia selectivity in vitro using EMT-6 mouse mammary carcinoma cells. Rodent biodistribution and small animal PET images were obtained from BALB/c mice implanted with EMT-6 tumors. One alkylated thiosemicarbazone was synthesized and examined. Results Of the three bis(selenosemicarbazone) ligands synthesized and examined, only 64Cu-diacetyl-bis(selenosemicarbazone) [ 64Cu-ASSM] was isolated in high-enough radiochemical purity to undertake cell uptake experiments where uptake was shown to be independent of oxygen concentration. The bis(thiosemicarbazone) complex synthesized, 64Cu-diacetyl-bis( N 4-ethylthiosemicarbazone) [ 64Cu-ATSE], showed hypoxia selectivity similar to 64Cu-ATSM although at a higher oxygen concentration. Biodistribution studies for 64Cu-ASSM and 64Cu-ATSE showed high tumor uptake at 20 min ( 64Cu-ASSM, 10.33±0.78% ID/g; 64Cu-ATSE, 7.71±0.46% ID/g). PET images of EMT-6 tumor-bearing mice visualized the tumor with 64Cu-ATSE and revealed hypoxia selectivity consistent with the in vitro data. Conclusion Of the compounds synthesized, only 64Cu-ASSM and 64Cu-ATSE could be examined in vitro and in vivo. Although the stability of bis(selenosemicarbazone) complexes increased upon addition of methyl groups to the diimine backbone, the fully alkylated species, 64Cu-ASSM, demonstrated no hypoxia selectivity. However, the additional alkylation present in Cu-ATSE modifies the hypoxia selectivity and in vivo properties when compared with Cu-ATSM.

Microenvironmental regulation of proliferation in multicellular spheroids is mediated through differential expression of cyclin-dependent kinase inhibitors.

Multicellular spheroids composed of transformed cells are known to mimic the growth characteristics of tumors and to develop gradients in proliferation with increasing size. This progressive accumulation of quiescent cells is presumably an active process that occurs in response to the microenvironmental stresses that develop within the three-dimensional structure, and, yet, little is known regarding either the signals that induce the cell cycle arrest or the molecular basis for the halt in proliferation. We have previously reported that regulation of cyclin-dependent kinase (CDK) inhibitors (CKIs) differs in monolayer versus spheroid cell culture. In this study, we have examined the expression of three CKIs in EMT6 mouse mammary carcinoma and MEL28 human melanoma spheroids, as a function both of spheroid size and of location within the spheroid. We report that expression of the CKIs p18(INK4c), p21(waf1/cip1), and p27(Kip1) all increase as the spheroid grows and develops a quiescent cell fraction. However, by examining protein expression in discrete regions of the spheroid, we have found that only p18(INK4c) and p27(Kip1) expression positively correlate with growth arrest, whereas p21(waf1/cip1) is expressed predominantly in proliferating cells. Further analysis indicated that, in the quiescent cells, p18(INK4c) is found in increasing association with CDK6, whereas p27(Kip1) associates predominantly with CDK2. In MEL28 cells, CDK2 activity is completely abrogated in the inner regions of the spheroid, whereas in EMT6 cells, CDK2 activity decreases in accordance with a decrease in expression. We also observed a decrease in all cell cycle regulatory proteins in the innermost spheroid fraction, including CDKs, CKIs, and cyclins. Induction of CKIs from separate families, as well as their association with distinct target CDKs, suggests that there may be multiple checkpoints activated to ensure cell cycle arrest in non-growth-conducive environments. Furthermore, because very similar observations were made in both a human melanoma cell line and a mouse mammary carcinoma cell line, our results indicate that these checkpoints, as well as the signal transduction pathways that activate them, are highly conserved.

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.

Reductive activation of doxorubicin by xanthine dehydrogenase from EMT6 mouse mammary carcinoma tumors

The role of enzymes in the reductive activation of various chemotherapeutic agents is an area of considerable interest in studies to better understand the selective toxicities of these agents. Xanthine dehydrogenase (XDH) is an enzyme capable of reductive activation of chemotherapeutic agents. Previously, this enzyme has not been extensively studied because of difficulties in its isolation. We recently isolated this enzyme from EMT6 mouse mammary carcinoma cells and showed that this enzyme is capable of activating mitomycin C. In this study, we examined whether XDH could activate the clinically important antineoplastic agent, doxorubicin. Drug activation was determined under aerobic and hypoxic conditions and at various pHs in order to simulate the different environments found in solid tumors. The results of these studies show that XDH reacts with doxorubicin via a two-electron reduction. This reduction is different from the modified and more extensively studied form of the enzyme, xanthine oxidase (XO), which reacts with doxorubicin via a one-electron reduction. Under hypoxic conditions, the formation of large quantities of 7-deoxydoxorubicin aglycone, a deactivation product of doxorubicin metabolism, may serve to moderate doxorubicin's antineoplastic activity. Under aerobic conditions, however, XDH activation led to a greater rate of formation of oxygen radicals than XO thereby possibly potentiating doxorubicin's cytotoxicity to aerobic tumor cells. Kinetic constants were determined for doxorubicin activation by XDH.

CAI: effects on cytotoxic therapies in vitro and in vivo.

CAI (NSC 609974; L651582), a new agent that has demonstrated antimetastatic activity in vitro and in vivo, was not very cytotoxic toward EMT-6 mouse mammary carcinoma cells in culture or toward FSaIIC fibrosarcoma cells in vivo. Coexposure of EMT-6 cells to CAI and antitumor alkylating agents under various environmental conditions did not markedly increase the cytotoxicity of cisplatin (CDDP), melphalan, or carmustine (BCNU). However, the combination of CAI and 4-hydroperoxycyclophosphamide (4-HC) produced much greater than additive killing of EMT-6 cells. CAI also increased the sensitivity of hypoxic EMT-6 cells to X-rays. CAI increased the cytotoxicity of cyclophosphamide toward FSaIIC tumor cells when animals were treated with single doses of both drugs. The effect of CAI on tumor cell killing by cyclophosphamide was greatest at high doses of the antitumor alkylating agent. CAI administration appeared to result in increased serum levels of prostaglandin E2 and leukotriene B4 in animals bearing the Lewis lung tumor. Administration of CAI on days 4-18 did not alter the growth of the Lewis lung carcinoma but did result in an increase in the tumor-growth delay produced by treatment with CDDP, cyclophosphamide, melphalan, BCNU, and fractionated radiation. Although CAI did not reduce the number of lung metastases present in Lewis lung carcinoma-bearing mice on day 20, it did appear to reduce the number of large (vascularized) metastases present on that day.

CYCLOOXYGENASE INHIBITORS - INVITRO AND INVIVO EFFECTS ON ANTITUMOR ALKYLATING-AGENTS IN THE EMT-6 MURINE MAMMARY-CARCINOMA

The nonsteroidal antiinflammatory drugs that inhibit cyclooxygenase block the formation of prostanoids in vivo. These agents may be useful as modulators of cytotoxic anticancer therapies. EMT-6 mouse mammary carcinoma cells growing in culture were exposed for 1 h or 24 h to eleven different nonsteroidal antiinflammatory agents or acetaminophen. None of these drugs was very cytotoxic. A concentration of 50muM of the nonsteroidal antiinflammatory drugs or acetaminophen was chosen for modulator combination studies with the antitumor alkylating agents CDDP, L-PAM, BCNU and 4-HC in cell culture. Several of the modulators protected the EMT-6 cells from the cytotoxicity of the antitumor alkylating agents; however, diflunisal, sulindac, indomethacin, acetaminophen and in some cases ibuprofen and tolmetin were positive modulators of the antitumor alkylating agents under the cell culture conditions tested. EMT-6 tumor cell survival studies and bone marrow CFU-GM survival studies were carried out with seven of the modulators and various doses of cyclophosphamide. Tolmetin, ibuprofen, sulindac, piroxicam and diflunisal in combination with cyclophosphamide produced increased tumor cell killing compared with cyclophosphamide alone without marked changes in toxicity to the bone marrow derived CFU-GM. In EMT-6 tumor growth delay experiments, none of the six modulators tested affected the growth of the tumors; however, tolmetin, ibuprofen, diflunisal and sulindac increased the tumor growth delay obtained with standard dose-schedules of cyclophosphamide or CDDP. When minocycline, a collagenase inhibitor, was added to treatment regimens including diflunisal or sulindac and either cyclophosphamide, CDDP or L-PAM further increases in tumor growth delay were obtained especially when L-PAM was the cytotoxic therapeutic agent. The number of lung metastases and the percentage lung metastases with diameters >3 mm were reduced by treatment with the modulator combinations alone and further reduced with the addition of the antitumor alkylating agents. These results indicate that agents which inhibit signaling pathways among tumor cells and between tumor cells and normal cells can be useful additions to cytotoxic therapies.

Bioactivation of mitomycin C by xanthine dehydrogenase from EMT6 mouse mammary carcinoma tumors.

Mitomycin C is an antineoplastic antibiotic requiring bioactivation to an alkylating species or to an intermediate capable of generating oxygen radicals for its toxic effect. The enzymes responsible for the in vivo activation of mitomycin C have been proposed to include NADPH-cytochrome-c reductase, DT-diaphorase, and xanthine oxidase. In this study, xanthine dehydrogenase, an enzyme structurally similar to xanthine oxidase, was assessed for its ability to activate mitomycin C. Partially purified xanthine dehydrogenase, from EMT6 mouse mammary tumors, was investigated for its ability to bioactivate mitomycin C under both aerobic and hypoxic conditions. We conducted this analysis by measuring mitomycin C-induced oxygen consumption, alkylating potential, and mitomycin C consumption and metabolite formation as determined by high-pressure liquid chromatography analysis. Bioactivation of mitomycin C by xanthine dehydrogenase under both aerobic and hypoxic conditions gave rise to the formation of a metabolite, 2,7-diaminomitosene. Formation of this metabolite and alkylating ability were greater under hypoxic than under aerobic conditions and were increased when the pH was decreased from 7.4 to 6.0. Mitomycin C consumption was the same under both aerobic and hypoxic conditions and was independent of pH. Oxygen consumption studies showed that xanthine dehydrogenase-activated mitomycin C consumed oxygen at a much lower rate than xanthine oxidase-activated mitomycin C. Xanthine dehydrogenase-activated mitomycin C appears to be a good alkylating species but a relatively poor generator of reactive oxygen when compared with xanthine oxidase activation under aerobic conditions. Xanthine dehydrogenase may play an important role in the bioactivation of mitomycin C to an alkylating species under both aerobic and hypoxic conditions.

Oxygen dependence of hypericin-induced phototoxicity to EMT6 mouse mammary carcinoma cells.

The photodynamic effect of hypericin on EMT6 mouse mammary carcinoma cells was investigated in vitro under aerobic and hypoxic conditions. Under aerobic conditions, hypericin-induced photocytotoxicity was dose dependent within a 1-50 microM range. Under hypoxic conditions, cells were resistant to hypericin-induced phototoxicity. In the dark, no cytotoxicity was observed at any hypericin concentration tested either aerobically or hypoxically. Cellular accumulation of hypericin, examined by chemical extraction and spectroscopy, occurred under both hypoxic and aerobic conditions. Fluorescence photomicrographs of cells exposed to hypericin corroborate drug uptake in the plasma membrane and subcellular regions. Our results demonstrate that hypericin cytotoxicity to EMT6 mouse mammary carcinoma cells in vitro is both light and oxygen-dependent. These results suggest that EMT6 cell kill caused by photoactivated hypericin is mediated by an oxygen-dependent mechanism, rather than by a type I oxygen-independent mechanism.

Induction of DNA‐Protein Crosslinks in Melanotic Cloudman S91 Mouse Melanoma Cells and EMT6 Mouse Mammary Carcinoma Cells by Monochromatic 254 and 405 nm Light

The rates of induction of DNA-protein crosslinks (DPC) by monochromatic radiation at 254 and 405 nm were compared in melanotic S91 mouse melanoma cells and EMT6 mouse mammary carcinoma cells. At 254 nm, the rates of induction of DPC are the same in the two cell lines, whereas, at 405 nm, the rate of induction of DPC in the melanotic cells is considerably less than that in the nonmelanotic cells. Since the major difference in the two cell lines with respect to absorption is melanin, the latter finding implies that intracellular melanin can protect against this DNA damage caused by a component of environmental carcinogenic solar radiation.

Chemotherapeutic attack of hypoxic tumor cells by the bioreductive alkylating agent mitomycin C

Since the cure of solid tumors is limited by the presence of cells with low oxygen contents, we have approached the development of treatment regimens and of new drugs for these tumors by investigating agents which are preferentially bioactivated under hypoxia. Major emphasis has been directed at studying the mode of action of the mitomycin antibiotics, as bioreductive alkylating agents. Using primarily the EMT6 mouse mammary carcinoma as a solid tumor model, we have found that mitomycin C and porfiormycin are preferentially toxic to cells with low oxygen contents. The mitomycin analog BMY-25282 is more toxic to hypoxic cells than are mitomycin C and porfiromycin; however, unlike these antibiotics, BMY-25282 is preferentially toxic to well-oxygenated cells. With these three mitomycins, we have observed a correlation between cytotoxicity to hypoxic cells, the rate of generation of reactive products, and the redox potentials of the drugs. Investigations of the enzymes in EMT6 cells that could possibly activate mitomycin C have revealed that cytochrome P-450 and xanthine oxidase are not present in measurable quantities and therefore are not responsible for activation of mitomycin C. Activities representative of NADPH-cytochrome c reductase and DT-diaphorase are present in these neoplastic cells. Comparison of these enzymatic activities in EMT6, CHO, and V79 cells with the rate of generation of reactive products under hypoxia shows a direct correlation between these two parameters, but there is no quantitative correlation between these two parameters and the amount of cytotoxicity. Use of purified NADPH-cytochrome c reductase and inhibitors of this enzyme demonstrated that NADPH-cytochrome c reductase can activate mitomycin C, but that it is probably not the only enzyme participating in this bioactivation in EMT6 cells. The DT-diaphorase inhibitor dicoumarol was employed to show that this enzyme is not involved in the activation of mitomycin C to a cytotoxic agent. Instead, DT-diaphorase appears to metabolize mitomycin C to a nontoxic product. This property has been exploited to develop a new treatment regimen for solid tumors. Using X-rays to eliminate well oxygenated cells of a solid tumor implant of the EMT6 carcinoma, we have found that the combination of dicoumarol plus mitomycin C is more toxic to hypoxic tumor cells in vivo than mitomycin C alone. Furthermore, knowledge of the biochemical mechanism of mitomycin C activation permits a prediction of which tumors can best be treated with this combination of drugs by measuring enzymatic activities in biopsy specimens.