Epirubicin-[ Anti-HER 2 / neu ] Synthesized with an Epirubicin-( C 13-imino )-EMCS Analog : AntiNeoplastic Activity against Chemotherapeutic-Resistant SKBr-3 Mammary Carcinoma in Combination with Organic Selenium

Purpose: Discover the anti-neoplastic efficacy of epirubicin-(C13-imino)-[anti-HER2/neu] against chemotherapeuticresistant SKBr-3 mammary carcinoma and delineate the capacity of selenium to enhance it’s cytotoxic anti-neoplastic potency. Methods: In molar excess, EMCH was combined with epirubicin to create a covalent epirubicin-(C13-imino)EMCH-maleimide intermediate with sulfhydryl-reactive properties. Monoclonal immunoglobulin selective for HER2/neu was then thiolated with 2-iminothiolane at the terminal ε-amine group of lysine residues. The sulfhydryl-reactive epirubicin-(C13-imino)-EMCH intermediate was then combined with thiolated anti-HER2/neu monoclonal immunoglobulin. Western-blot analysis was utilized to characterize the molecular weight profiles while binding of epirubicin-(C13-imino)[anti-HER2/neu] to membrane receptors was determined by cell-ELISA utilizing populations of SKBr-3 mammary carcinoma that highly over-expresses HER2/neu complexes. Anti-neoplastic potency of epirubicin-(C13-imino)-[anti-HER2/ neu] between the epirubicin-equivalent concentrations of 10 M and 10 M was determined by vitality staining analysis with and without the presence of selenium (5 μM). Results: Epiribucin-(C13-imino)-[anti-HER2/neu] between epirubicin-equivalent concentrations of 10 M to 10 M consistently evoked higher anti-neoplastic potency than “free” nonconjugated epirubicin which corresponded with previous investigations utilizing epirubicin-(C3-amide)-[anti-HER2/neu] and epirubicin-(C3-amide)-[anti-EGFR]. Selenium at 5 mM consistently enhanced the cytotoxic anti-neoplastic potency of epirubicin-(C13-imino)-[anti-HER2/neu] at epirubicin equivalent concentrations (10–12 to 10–7 M). Conclusions: Epirubicin-(C13-imino)-[anti-HER2/neu] is more potent than epirubicin against chemotherapeutic-resistant SKBr-3 mammary carcinoma and selenium enhances epirubicin-(C13-imino)-[anti-HER2/neu] potency. The methodology applied for synthesizing epirubicin-(C13-imino)-[anti-HER2/neu] is relatively time convenient and has low instrumentation requirements.


Introduction
Monoclonal immunoglobulin fractions with binding avidity for HER2/neu and EGFR have demonstrated effectiveness in the treatment of mammary carcinoma and other neoplastic disease states that over-express these trophic membrane-associated receptors.Unfortunately, immunoglobulin-based therapeutics of this type reportedly have an inability to exert significant cytotoxic activity or completely resolve neoplastic conditions [1][2][3][4][5][6][7] unless they are applied in combination with chemotherapy or other forms of anti-cancer treatment [8,9].Despite general familiarity with the influence of anti-HER2/neu immunoglobulin on cancer cell biology and its applica-Epirubicin-[Anti-HER2/neu] Synthesized with an Epirubicin-(C -imino)-EMCS Analog: Anti-Neoplastic Activity 23 13 Against Chemotherapeutic-Resistant SKBr-3 Mammary Carcinoma in Combination with Organic Selenium tion in clinical oncology there is surprisingly little known about covalent anthracycline-[anti-HER2/neu] immunochemotherapeutics and their potential to exert enhanced levels of anti-neoplastic activity against chemotherapeutic-resistant mammary carcinoma [10].
A small collection of organic chemistry reactions can be used to covalently link anthracycline-class chemotherapeutic agents to monoclonal immunoglobulin or other biologically active protein fractions.One common methodology for the synthesis of anthracycline conjugates involves the creation of a covalent amide bond at the single C 3 α-monoamine group of the anthracycline carbohydrate moiety [10][11][12][13].Dual combinations of epirubicin-(C 3 -amide)-[anti-HER2/neu] and epirubicin-(C 3 -amide)-[anti-EGFR] have demonstrated synergistic levels of anti-neoplastic activity against chemotherapeutic-resistant SKBr-3 mammary carcinoma which is complemented by the simultaneous application of competitive P-glyco-protein inhibitors [10].
Immunochemotherapeutics synthesized through the creation of a covalent imino bond at the C 13 -keto group of anthracyclines have been described [27][28][29] utilizing only a rather limited spectrum of monocloncal immunoglobulin fractions.Furthermore, in contrast to immunoglobulin-based diagnostic radiopharmaceuticals and radioimmunotherapeutics, there are few descriptions of the molecular design, synthesis and efficacy evaluation of covalent anthracycline immunochemotherapeutics that exert selective anti-neoplastic properties against mammary carcinoma propagated in-vitro in tissue culture [30,31], in-vivo xenografts [32], or natural clinical disease states.Immunochemotherapeutics synthesized as anthracycline (C 13 -imino)-immunoglobulin with selective "targeted" delivery properties for breast cancer have predominately utilized anti-Lewis Y antigen monoclonal immunoglobulin (e.g.BR96/SGN15) [32][33][34][35] in part because it has cross-cancer-efficacy against lung carcinoma [32,35,36], intestinal carcinoma [32][33][34]37], and ovarian carcinoma [33].The full anti-neoplastic efficacy of covalent epirubicin-(C 13 -imino)-[anti-HER2/neu] immunochemotherapeutics proportedly increased through enhanced acid-sensitive mediated liberation of the anthracycline within the micro-environment of the phagolysosome/endolysosome (pH 5.0 -5.5) remains to be delineated.Similarly, although previous investigations have characterized the influence of selenium on the biology of various neoplastic cell type, little is know about the potential for selenium to enhance the cytotoxiic anti-neoplastic potency of epirubicin-(C 13 -imino)-[anti-HER2/neu] against chemotherapeutic-resistant mammary carcinoma.
Phase-II: Synthesis of Epirubicin-(C 13 -imino)-EMCH Sulfhydryl Reactive Intermediate-The C 13 -keto group of epirubicin (1.479 × 10 -2 mg, 2.55 × 10 -5 mMole in methanol) was reacted with the hydrazide group of the heterobifunctional covalent cross-linking reagent, N-εmaleimi-docaproic acid hydrazide in the presence of trifluoroacetic acid (EMCH: 43.2 μg, 1.275 × 10 -4 mmol in methanol) and then incubated at 25˚C for 96 hours in concert with constant gentle stirring [17,24].Recrystalization of epirubicin-(C 13 -imino)-EMCH to remove residual unreacted EMCH was performed by the addition of acetonitrile until a slight opalescent appearance de-veloped followed by incubation at -20˚C for 24 hours.Recrystalized epirubicin-EMCH was harvested by centrifugation and rinsed in cold acetonitrile.The resulting recrystalization supernatant was partially evaporated under a stream of nitrogen (N 2 ) gas in order to maximize yield of total epirubicin-(C 13 -imino)-EMCH product.Prior to Phase II synthesis procedures, residual methanol in aliquots of recrystalized epirubicin-EMCH was removed under a gentle stream of nitrogen gas and then re-dissolved in dimethylsulfoxide (DMSO).

Synthesis Methodology II: Epirubicin-(C 3 -amide)-SMCC-Immunoglobulin
Phase-I: Introduction of Sulfhydryl Groups into Immunoglobulin at Lysine ε-Amine Groups-Monoclonal Immunoglobulins with selective binding avidity against human epidermal growth factor receptor type 1 (EGFR) and HER2/neu (ErbB-2, CD 340) were acquired as desiccated preparations in 1.5 mg amounts.Simultaneous removal of xylose and buffer-exchange into PBS (phosphate 0.1 M, NaCl, 0.15 M, pH 7.3) was performed prior to semi-synthesis procedures using micro-scale "desalting" column chromatography resulting in a final IgG concentration of 13.3 μM (> 2.0 mg/ml in 700 μl).Individual IgG monoclonal antibodies at a concentration of approximately 2.0 mg/ml in 700 μl of PBS where combined with N-succinimidyl-S-acetylthioacetate (SATA) at a molar ratio of 1:9 (3 μl of a 55 mM SATA formulation in DMSO).Preparations were incubated at 25˚C for 30 minutes and then buffer exchanged into PBS (phosphate 0.1 M, NaCl 0.15 M, pH 7.3) using micro-scale "desalting" column chromatography.
Phase-III: Covalent Incorporation of Epirubicin-(C 3amide)-SMCC at SATA Modified Immunoglobulin Lysine ε-Amine Groups-Immediately prior to Phase-III synthesis methods, SATA-IgG preparations were deacetylated (activated) with hydroxylamine (0.5 M with EDTA 25 mM in PBS, pH 7.3) at a 10:1 volumetric ratio for 2 hours with continual stirring at 25˚C thereby generating a primary sulfhydryl group.Residual unreacted SATA was removed from MoAb IgG preparations by buffer exchange into PBS-EDTA (phosphate 0.1, NaCl 0.15 M, EDTA 10 mM, pH 7.3) using micro-scale "desalting" column chromatography.Sulhydryl content was subsequently determined using an Ellman's Reagent based assay system.
The primary sulfhydryl group of deacetylated SATA-IgG preparations was subsequently reacted with the maleimido group of SMCC-epirubicin followed by incubation at 25˚C with continual stirring for 30 minutes.Residual epirubicin was removed from covalent epirubicin immunochemotherapeutic preparations by a buffer exchange into PBS (phosphate 0.1, NaCl 0.15 M, EDTA 10 mM, pH 7.3) using micro-scale "desalting" column chromatography.

Analysis, Characteristics and Properties
General Analysist-Determination of the IgG concentration within covalent epirubicin-[anti-HER2/neu] immunochemotherapeutics was determined by measuring absorbance at 280 nm.Measurement of epirubicin concentrations was established by excitation at 485 nm and measurement of emission at 538 nm using known concentrations of epirubicin to generate a standard reference control curve sufficient to generate a linear equation for determination of epirubicin-equivalent concentrations between 10 -9 M and 10 -5 M. Determination of the non-conjugated "free" epirubicin concentration contained in covalent epirubicin immunochemotherapeutic preparations was established by chloroform extraction [19,41,42], with the organic phase collected by pipette, evaporated to dryness under a stream of nitrogen gas, and the resulting residue dissolved in Tris buffered saline (50 mM, pH 7.4) prior to further analysis.Adjusted epirubicin:immunoglobulin molar incorporation indexes were calculated by measuring absorbance at 485 nm and 280 nm respectively and by correcting for absorbance from epirubicin at 280 nm.
Molecular Mass-Dependent Separation of Covalent Epirubicin Immunochemotherapeutics by Non-Reducing SDS-PAGE-Covalent epirubicin-[anti-HER2/neu] immunochemotherapeutics in addition to a anti-HER2/neu immunoglobulin reference control fraction were adjusted to a standardized protein concentration of 60 μg/ml and then combined 50/50 v/v with conventional SDS-PAGE sample preparation buffer (Tris/glycerol/bromphenyl blue/ SDS) formulated without 2-mercaptoethanol or boiling.The epirubicin immunochemotherapeutics, a reference control anti-HER2/neu immunoglobulin fraction (0.9 μg/well) and a mixture of pre-stained molecular weight markers were then developed by non-reducing SDS-PAGE (11% acrylamide) performed at 100 V constant voltage at 3˚C for 2.5 hours.
Western-Blot Immunodetection Analyses-Covalent epirubicin-[anti-HER2/neu] immunochemotherapeutics following mass-dependent separation by non-reducing SDS-PAGE were equilibrated in tank buffer devoid of methanol.Mass-separated epirubicin-[anti-HER2/neu] immunochemotherapeutics contained in acrylamide SDS-PAGE gels were then laterally transferred onto sheets of nitrocellulose membrane at 20 volts (constant voltage) for 16 hours at 2˚C to 3˚C with the transfer manifold packed in crushed ice.
Potential for selenium to enhance the anti-neoplastic potency of epirubicin-[anti-HER2/neu] against SKBr-3 mammary carcinoma was determined by incubating cell populations with methylseleninate (CH 3 SeO 2 H at 5 μM or 0.635 μg/ml) [43][44][45][46] at a concentration pre-determined to not promote a markedly detrimental influence on cell vitality.Epirubicin immunochemotherapeutics formulated with or without selenium where then incubated with monolayer populations of SKBr-3 mammary carcinoma for 72-hours at 37˚C under a gas atmosphere of air (95%) and carbon dioxide (5% CO 2 ).Complementary investigations evaluated the influence of increasing selenium concentrations (5 μM to 50 μM) in combination with a fixed epirubicin concentration (10 -8 M), in addition to increasing epirubicin concentration (10 -12 M to 10 -7 M) in combination with a fixed selenium concentration (45 mM) over a 72-hour incubation period with chemotherapeutic-resistant SKBr-3 mammary carcinoma.Lastly, as a point of reference, selenium cytotoxic anti-neoplatic activity was compared to equal micro-molar concentrations of celecoxib in chemotherapeutic-resistant SKBr-3 mammary carcinoma.
The contents of 96-well microtiter plates at 72-hours were removed manually by pipette and SKBr-3 mammary carcinoma monolayers serially rinsed (n = 3) with PBS followed by incubation with MTT vitality stain reagent (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide 5mg/ml) formulated in RPMI-1640 growth media devoid of pH indicator or bovine fetal calf serum.During an incubation period of 3 hours at 37˚C under a gas atmosphere of air (95%) and carbon dioxide (5% CO 2 ) the enzyme mitochondrial succinate dehydrogenase was allowed to convert MTT vitality stain to navy-blue formazone crystals.Contents of the 96-well microtiter plate were then removed, and serially rinsed with PBS (n = 3) followed by dissolving of the resulting blue intracellular formzone crystals with DMSO (300 μl/well).Spectrophotometric absorbance of the navy-blue colored supernatant was then measured at 570 nm using a computer integrated microtiter plate reader.

Results
In Phase I of the synthesis method, the C 13 -keto group of epirubicin was reacted with the hydrazide group of N-ε-maleimidocaproic acid hydrazide (EMCH) to create an imino bond resulting in the formation of a covalent epirubicin-(C 13 -imino)-EMCH intermediate (Figure 1).In Phase II of the synthesis method, the thiol-reactive maleimide group of the epirubicin-(C 13 -imino)-EMCH intermediate was covalently linked to sulfhydryl groups introduced at the ε-amine of lysine amino acid residues within HER2/neu immunoglobulin fractions following thiolation with 2-iminothiolane (2-IT) reagent (Figure 1).The anthracycline molar incorporation index for epirubicin-(C 13 -imino)-[anti-HER2/neu] was 40% while epirubicin-(C 3 -amide)-[anti-HER2/neu] and epirubicin-(C 3 -amide)-[anti-EGFR] were produced at a level of 27.5% and 40.7% respectively utilizing essentially identical conditions [10].The percent of non-covalently bound anthracycline contained in covalent epirubicin immunochemotherapeutics following the application of micro-scale desalting/buffer exchange column chromatography was consistently < 4.0% where residual non-covalently bound anthracycline generally can not be removed by performing serial/repeated separations by column chromatography) [23].Higher epirubicin molar incorporation indexes are possible to achieve with modifications in the synthesis methodology but the harsher conditions required for such purposes are accompanied by substantial reductions in the final yield of covalent immunochemotherapeutic [47], and declines in antigen-immunoglobulin binding affinity (e.g.cell-ELISA analysis).
Covalent epirubicin-[anti-HER2/neu] immunochemotherapeutics mass-separated by SDS-PAGE and developed by Western-blot immunodetection analysis in combination with chemiluminescent autoradiography detected a condensed 150-kDa band between a 5.0-kDa to 450-kDa molecular weight range (Figure 2) The molecular weight of 150-kDa detected for epiribucin-[anti-HER2/neu] immunochemotherapeutics directly corresponded with the molecular weight/mass detected for the anti-HER2/neu immunoglobulin reference control fraction and the known molecular weight for immunoglobulin (Figure 2).Analogous results have been reported for similar covalent immunochemotherapeutics [10,14,48].
Cytotoxicity Analysis-The anti-neoplastic potency of Against Chemotherapeutic-Resistant SKBr-3 Mammary Carcinoma in Combination with Organic Selenium  epirubicin concentration (10 -12 M to 10 -7 M) in combination with a fixed selenium concentration (45 mM) additively increased total cytotoxic anti-neoplastic potency exerted against chemotherapeutic-resistant SKBr-3 mammary carcinoma (Figure 8).When utilized as a combination, epirubicin and selenium exerted progressive increases in cytotoxic anti-neoplastic potency where a detectable threshold effect was observed individually with either epirubicin or selenium (Figures 7 and 8).Compared to celecoxib, micro-molar equivalent concentrations of selenium exerted higher cytotoxic anti-neoplastic potency against chemotherapeutic resistant SKBr-3 mammary carcinoma either alone or in combination with epirubicin chemotherapeutic (Figure 9).Individual fractions of anti-HER2/neu and anti-EGFR monoclonal immunoglobulin alone did not exert any detectable cytotoxic anti-neoplastic activity against SKBr-3 mammary carcinoma at 72-hours (Figure 7) which is in accord with previous investigations [10,48,[50][51][52][53].

Discussion
One proposed attribute of covalent anthracycline immunochemotherapeutics synthesized utilizing a reactive hydrazide is that it creates a covalent but "acid-sensitive"
The acid-sensitive properties of anthracycline-(C 13imino) immunochemotherapeutics generated through the
Cell Binding-Total membrane bound IgG profiles in SKBr-3 mammary carcinoma populations for epirubicin-(C 13 -imino)-[anti-HER2/neu] proportionally increased with elevations in total immunoglobulin (Figure 3).Seemingly modest alterations in synthesis chemistry and elevations in chemotherapeutic molar incorporation index can profoundly influence immunoglobulin binding properties [53].Given this perspective, the relatively mild conditions employed during the course of synthesis procedures and relatively modest 40% molar incorporation index collectively contributed to the high biological integrity of epirubicin-(C 13 -imino)-[anti-HER2/neu] based on results from SDS-PAGE/immunodetection and cell-ELISA analyses.Relatively higher anthracycline immunoglobulin molar incorporation indexes can be attained by implementing harsher synthesis conditions.Such modifications often result in only modest declines in immunoreactivity (e.g.86% for a 73:1 ratio) but disproportionate declines in anti-neoplastic activity down to potency levels substantially lower than those measured for non-conjugated "free" anthracycline [60].Internalization of covalent epirubicin-[anti-HER2/neu] and epirubicin-[anti-EGFR] immunochemotherapeutics following binding to HER2/neu and EGFR receptors over-expressed by SKBr-3 mammary carcinoma occurs predominately by mechanisms of receptor-mediated endocytosis analogous to previous descriptions for similar covalent anthracycline immunochemotherapeutics [59].Although specific data for SKBr-3 HER2/neu and EGFR receptor complexes is limited [10], other neoplastic cell lines like metastatic multiple myeloma are known to internalize and metabolize approximately 8 × 10 6 molecules of anti-CD74 monoclonal antibody per day [61].
Several modifications in analytical methodology could have been implemented to substantially increase the level of anti-neoplastic activity exerted by epirubicin-(C 13imino)-[anti-HER2/neu] and epirubicin-(C 3 -amide)-[anti-HER2/neu].Cytotoxic anti-neoplastic potency could have alternatively been assessed with a breast cancer cell type that was not chemotherapeutic-resistant similar to populations utilized to evaluate majority of the covalent biochemotherapeutics reported in the literature to date.Rare exceptions in this regard have been the evaluation of anti-chondroitin sulfate proteoglycan 9.2.27 surface marker daunorubicin conjugates (against chemotherapeutic-resistant M21 metastatic melanoma) [50,53,68]; anthracycline conjugates of epidermal growth factor (EGF) or EGF fragment (evaluated for their anti-neoplastic potency against chemotherapeutic-resistant MCF-7AdrR mammary carcinoma) [69]; and epirubicin-[anti-HER2/ neu] or epirubicin-[anti-EGFR] potency (assessed against chemotherapeutic-resistant SKBr-3 mammary carcinoma) [10].Similarly, the cytotoxic anti-neoplastic potency of covalent epirubicin-[anti-HER2/neu] or epirubicin-[anti-EGFR] immunochemotherapeutics would likely have been higher if it had been accessed in-vivo since many anti-cancer immunochemotherapeutics produce much greater cytotoxic activity in-vivo compared to their in-vitro level of potency [62,70].Enhanced levels of cytotoxic anti-neoplastic activity of covalent epirubicin immunochemotherapeutics can presumably be attained at least in part by several host mechanisms that can not be effec-tively assessed in-vitro in a tissue culture environment.Relevant examples in this regard include, 1) potentially additive or synergistic anti-neoplastic activity achieved through dual chemotherapeutic/anti-trophic receptor immunoglobulin and dual anti-trophic receptor/anti-trophic receptor immunoglobulin combinations (Figure 10) [10]; 2) antibody/antigen/complement complex induced cytolysis; and 3) antibody-dependent cell-mediated immune responses.Lastly, analytical methodologies could have been modified to detect cytotoxic anti-neoplastic potency in chemotherapeutic-resistant SKBr-3 mammary carcinoma in a more sensitive manner than is possible with MTT vitality/proliferation stain assay systems.Instead, declines in viability could have been measured by employing either the [H 3 ]-thymidine cell proliferation assay, or ATP-based assay since they reportedly are ≥ 10-fold more sensitive than MTT vitality stain assays [71,72].Despite this consideration, measurement of cell vitality utilizing MTT reagent based assays have, and continue to be extensively employed for the routine assessment of chemotherapeutic anti-neoplastic activity [73][74][75][76].However, it is important to note that the creation of lethal injury detected with MTT reagent based assays usually Against Chemotherapeutic-Resistant SKBr-3 Mammary Carcinoma in Combination with Organic Selenium occurs at higher chemotherapeutic doses than those required to produce alteration in transcription/translation, or [H 3 ]-thymidine and ATP-based assay systems.Selenium in simultaneous combination with epirubicin-(C 13 -imino)-[anti-HER2/neu] was analyzed to determine if this element enhances the cytotoxic anti-neoplastic potency of covalent anthracyline immunochemotherapeutics against chemotherapeutic-resistant SKBr-3 mammary carcinoma (Figure 6).Consistently, selenium (methylseleninate 5 mM fixed concentration) additively enhanced [77] the cytotoxic anti-neoplastic potency of epirubicin-(C 13 -imino)-[anti-HER2/neu] formulated at anthracycline-equivalent concentrations between 10 -12 M to 10 -7 M, but this effect was not statistically significant (Figure 6).Alterations were most pronounced for epirubicin-(C 13 -imino)-[anti-HER2/neu] at epirubicin-equivalent concentrations < 10 -7 M where vitality/prolif-eration profiles implied that the cytotoxic anti-neoplastic activity detected at 10 -7 M was almost entirely evoked through liberation of the anthracycline moiety.When the epirubin equivalent concentration was fixed at 10 -8 M, gradient increases in selenium concentration from 5 mM to 50 mM produced significantly higher levels of cytotoxic activity that observed for epirubicin alone (Figure 7).Since the results for both epirubicin-(C 13 -imino)-[anti-HER2/neu] and epirubicin at 10 -8 M with and without the presence of selenium at 5 mM were not significant different, it is fully anticipated that compared to epirubicin, highly analogous results would have been detected for epirubicin-(C 13 -imino)-[anti-HER2/neu] formulated at epirubicin equivalent concentrations of 10 -8 M in combination with or without selenium at final concentrations of 5 mM and 50 mM.In the interpretation of this latter consideration, it is important to keep in perspective that the cytotoxic activity of epirubicin-(C 13 -imino)-[anti-HER2/neu] is greater than epirubicin at epirubicinequivalent concentrations (Figure 4) Selenium therefore can potentially be utilized in combination with lower epirubicin-equivalent concentrations of epirubicin-(C 13imino)-[anti-HER2/neu] or epirubicin to attain additive increases in cytotoxic anti-neoplastic potency (Figures 6,  7 and 8).Interestingly, selenium exerts greater cytotoxic anti-neoplastic activity compared to celecoxib [78][79][80][81] when analyzed at micro-molar equivalent concentrations (Figure 9).
Serum selenium concentrations provide prognostic implications because they can serve as a positive indicator for predicting effective dosage levels, optimum delivery routes, treatment response, and long-term survival [82].The biology of cancer cells is modified by selenium based on it's capacity to 1) induce apoptosis in doxorubicin-resistant lung small-cell carcinoma (selenite 10 μM) [46]; 2) promote severe ER stress (leukemia cell types) [83]; 3) reduce vitality of multidrug-resistant leukemia (selenitetriglycerides 10 μg/ml to 40 μg/ml) [84]; and 4) influence Fas signaling in MCF-7 mamary carcinoma in the presence of doxorubicin (methylseleninate 5 μM) [43].Relatively few investigations have previously described the comparative cytotoxic anti-neoplastic activity of multiple selenium analogs.
Enhancements in anthracycline anti-neoplastic potency in the presence of selenium coincides with increases in apoptosis [44,86] detected as increases in DNA fragmentation, declines in DNA synthesis, elevations in "round-cell" formation, membrane "blebbing" phenomenon and decreased N-(methylamino)-isobutyric acid uptake (MeAIB: non-metabolizable amino acid analog) [86].Each of these alterations is selenium dose-dependent whereby an optimal effect occurs at "free" elemental selenium concentrations between 4 ng/ml and 40 ng/ml over a 72-hour period [86].
Several biochemical mechanisms modified by selenium likely contribute to it's enhancement of anthracycline potency against different neoplastic cell types.Selenium (selenite) alone causes cell death through activation of the pro-apoptotic transcription factor GADD153 and in leukemia cells high concentrations promote p53 activation [83].Independently, selenium (selenite) mediates anti-neoplastic activity through p53 activation and increased oxidative stress which collectively precipitate mitochondrial dysfunction and caspase activation (leukemia cell types) [83].Selenium promotes apoptosis by also increasing Fas-associated death domain (FADD) expression and enhanced caspase-8 recruitment to Fas and FADD (MCF7 breast cancer) [43].Progressive increases in selenium concentrations (1 μM to 10 μM) induce dose-dependent increases in the amount and activity of thioredoxin reductase in non-resistant neoplastic cells, Against Chemotherapeutic-Resistant SKBr-3 Mammary Carcinoma in Combination with Organic Selenium but it precipitates declines in thioredoxin reductase in doxorubicin-resistant neoplastic cells (small cell carcinoma) [46].Functionally, thioredoxin reductase biochemically reduces thioredoxin responsible for mediating the final step of the electron-transfer pathway for nucleoside diphosphate reduction which is essential in cancer cells for growth and survival.
Selenium induces a number of effects within neoplastic cells that are known to increase anthracycline antineoplastic activity and potency.High selenium concentrations elevate oxidative stress [83] in part by reducing catalase enzyme activity (↓ H 2 O 2 → H 2 O + O 2 ) [85] that in turn complements doxorubicin-induced declines in peroxidase activity (↓ ROOR' + 2 e -+ 2H + → ROH + R'OH) [85].Cytotoxic synergy produced by selenium and anthracyclines combinations is further achieved through an elevated plane of apoptosis as a result of mitochondrial caspase-9 activation (MCF7 breast cancer cells) [43].Such alterations correlate with the detection of selenium-induced DNA strand breaks/fragmentation (leukemia) [84] that occur at a 2.5-fold greater level in the presence of selenium/doxorubicin combinations (B-cell lymphoma) [45].Selenium-mediated DNA fragmentation and strand breaks (e.g.selenium-induced apoptosis) develop secondary to increases in the formation or molecular stability of topoisomerase II-DNA complexes [88].Such a property is likely complemented by the greater capacity of anthracyclines to create oxygen "free" radical species in neoplastic cell types that have characteristically high [Fe +2 /Fe +3 ] cytosol concentrations, in addition to anthracycline binding to and inhibition of topoisomerase II.
The collective effect of selenium with anthracyclines on Akt is presently considered to be one of, if not the most important molecular mechanism responsible for the complementary anti-neoplastic activity exerted by this dual combination (MCF-7 breast cancer) [44].Selenium (selenite) at high concentrations suppresses doxorubicin-induced Akt phosphorylation [44] where Akt is a component of anti-apoptotic pathays [83] and influences glucose metabolism.Selenium is largely incapable of sensitizing cells to doxorubicin when Akt is constitutively activated (MCF-7 breast cancer cells) [44].Doxorubicin-induced increases in Akt phosphorylation occurs simultaneously with elevations in the Akt phosphorylated substrates phospho-GSK3-β (glycogen synthase kinase-3-beta) and phosphor-FOXO3a (forkhead box O3) which each lose pro-apoptotic properties post phosphorylation (MCF-7 breast cancer cells) [44].Selenium reduces phospho-GSK3beta abundance induced by doxorubicin, while chemical inhibition of GSK3beta biochemical activity mutes apoptotic responses created by selenium/doxorubicin combinations (MCF-7 breast cancer) [44].Selenium is additionally believed to trigger apoptosis by increasing FOXO3a transcriptional factor activity [44] which occurs in concert with upregulation of Bim [44] and PUMA, or down-regulation of FLIP anti-apoptotic protein.
Interesting, in a slightly different context, selenium and α-tocopherol may both potentially improve internalization of chemotherapeutic-ligand conjugates designed to selectively "target" over-expressed receptor complexes known to undergo receptor-mediated endocytosis [89].Such speculation is based on the observation that selenium and α-tocopherol deficiencies reduce receptor-mediated processes possibly due to greater levels of membrane oxidation and alterations in membrane fluidity.Selenium therefore holds promise as a form of adjunct therapy capable of increasing the cytotoxic anti-neoplastic potency of covalent anthracycline immunochemotherapeutics against aggressive and resistant forms of cancer.
The method described for the synthesis of epirubicin-(C 13 -imino)-[anti-HER2/neu] introduces a minimum number of cyclic ring structures into the final form of the covalent epirubicin-immunochemotherapeutic compared to other methodologies which has been proposed to minimize the induction of in-vivo humoral immune responses.Logistically, the scheme designed for the synthesis of epirubicin-(C 13 -imino)-[anti-HER2/neu] is relatively convenient to execute because the number of procedural steps required to adjust the pH of reaction buffers has been intentionally reduced [67].Complementary attributes included minimal instrumentation requirements Against Chemotherapeutic-Resistant SKBr-3 Mammary Carcinoma in Combination with Organic Selenium and the utilization of pre-sythesized (stock) sulfhydrylreactive maleimodocaproyl) hydrazone anthracycline (C 13 -imino) derivative in concert with thiolated anti-HER2/neu which increased convenience and minized time requirements [19,28,47,89].

Figure 7 . 8 M
Figure 7. Influence of increasing selenium concentrations on the cytotoxic anti-neoplastic potency of epirubicin (10 -8 M) measured as a function of chemotherapeutic-resistant SKBr-3 mammary carcinoma cell vitality.Legend: () selenium (methylseleninate); and () selenium (methylseleninate) in combination with epirubicin (10 -8 M).SKBr-3 monolayers were incubated with immunochemotherapeutics over a 72-hour period and cytotoxicity measured as a function of MTT cell vitality relative to matched negative reference controls.

Figure 8 .
Figure 8. Influence of increasing epirubicin concentrations on the cytotoxic potency of selenium (45 μM) measured as declines in the cellular vitality of chemotherapeutic-resistant SKBr-3 mammary carcinoma.Legend: () epirubicin chemotherapeutic in combination with selenium (methylseleninate 45 μM); and () epirubicin chemotherapeutic alone.SKBr-3 monolayers were incubated with immunochemotherapeutics over a 72-hour period and cytotoxicity measured as a function of MTT cell vitality relative to matched negative reference controls.

Figure 9 .
Figure 9. Relative cytotoxic anti-neoplastic potency of selenium and celecoxib measured as a function of chemotherapeutic-resistant SKBr-3 mammary carcinoma survivability with and without epirubicin (10 -7 M).Legend: () selenium (methylseleninate) 30 μM, 40 μM, and 50 μM with and without the presence of epirubicin at 10 -7 M fixed concentration; () celecoxib at 30 μM, 40 μM, and 50 μM with and without the presence of epirubicin at 10 -7 M fixed concentration.Monolayers of SKBr-3 mammary carcinoma were incubated with immunochemotherapeutics over a 72-hour period and cytotoxicity measured as a function of MTT cell vitality relative to matched negative reference controls.