Synergistic Anticancer Action of Lysosomal Membrane Permeabilization and Glycolysis Inhibition.

We investigated the in vitro and in vivo anticancer effect of combining lysosomal membrane permeabilization (LMP)-inducing agent N-dodecylimidazole (NDI) with glycolytic inhibitor 2-deoxy-d-glucose (2DG). NDI-triggered LMP and 2DG-mediated glycolysis block synergized in inducing rapid ATP depletion, mitochondrial damage, and reactive oxygen species production, eventually leading to necrotic death of U251 glioma cells but not primary astrocytes. NDI/2DG-induced death of glioma cells was partly prevented by lysosomal cathepsin inhibitor E64 and antioxidant α-tocopherol, suggesting the involvement of LMP and oxidative stress in the observed cytotoxicity. LMP-inducing agent chloroquine also displayed a synergistic anticancer effect with 2DG, whereas glucose deprivation or glycolytic inhibitors iodoacetate and sodium fluoride synergistically cooperated with NDI, thus further indicating that the anticancer effect of NDI/2DG combination was indeed due to LMP and glycolysis block. The two agents synergistically induced ATP depletion, mitochondrial depolarization, oxidative stress, and necrotic death also in B16 mouse melanoma cells. Moreover, the combined oral administration of NDI and 2DG reduced in vivo melanoma growth in C57BL/6 mice by inducing necrotic death of tumor cells, without causing liver, spleen, or kidney toxicity. Based on these results, we propose that NDI-triggered LMP causes initial mitochondrial damage that is further increased by 2DG due to the lack of glycolytic ATP required to maintain mitochondrial health. This leads to a positive feedback cycle of mitochondrial dysfunction, ATP loss, and reactive oxygen species production, culminating in necrotic cell death. Therefore, the combination of LMP-inducing agents and glycolysis inhibitors seems worthy of further exploration as an anticancer strategy.

Synthesis, identification and in vivo studies of tumor-targeting agent peptide doxorubicin (PDOX) to treat peritoneal carcinomatosis of gastric cancer with similar efficacy but reduced toxicity.

BACKGROUND: This work aimed to synthesize a cathepsin B (CTSB)-cleavable tumor-targeting prodrug peptide doxorubicin (PDOX) and study the in vivo efficacy and toxicities on an animal model of gastric peritoneal carcinomatosis (PC). METHODS: PDOX was synthesized using doxorubicin (DOX) attaching to a CTSB-cleavable dipeptide Ac-Phe-Lys and a para-amino-benzyloxycarbonyl (PABC) spacer. PC model was established by injecting VX2 tumor cells into the gastric sub-mucosa of 40 rabbits, which then were randomized into 4 groups: the Control (n = 10) without treatment, the HIPEC (n = 10) receiving cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC), the PDOX (n = 10) and the DOX (n = 10) receiving systemic chemotherapy with PDOX 50.0 mg/kg or DOX 5.0 mg/kg, respectively, after CRS + HIPEC. RESULTS: The median overall survivals (OS) were 23.0 d (95% CI: 19.9 d - 26.1 d) in the Control, 41.0 d (36.9 d - 45.1 d) in the HIPEC, 65.0 d (44.1 d - 71.9 d) in the PDOX, and 58.0 d (39.6 d - 54.4 d) in the DOX. Compared with the Control, the OS was extended by 70% in the HIPEC (p < 0.001) and further extended by 40% in the DOX (p = 0.029) and by 58% in the PDOX (p = 0.021), and the PC severity was decreased in the HIPEC and further decreased in the PDOX and DOX. Animals receiving DOX treatment showed hematological toxicities with marked reduction of white blood cells and platelets, as well as cardiac toxicities with significant increases in creatine kinase mb isoenzyme, evident myocardium coagulation necrosis, significant nuclear degeneration, peri-nucleus mitochondria deletion, mitochondria-pyknosis, and abnormal intercalated discs. But these toxicities were not evident in the PDOX. CONCLUSIONS: PDOX is a newly synthesized tumor-targeting prodrug of DOX. Compared with DOX, PDOX has similar efficacy but reduced hematological and cardiac toxicities in treating rabbit model of gastric PC.

Anti-cancer effects of novel doxorubicin prodrug PDOX in MCF-7 breast cancer cells.

Ac-Phe-Lys-PABC-DOX (PDOX) is a smart doxorubicin (DOX) prodrug designed to decrease toxicities while maintaining the potent anticancer effects of DOX. This study was aimed at elucidating the effectiveness and toxicities of DOX and PDOX in patient-derived MCF-7 breast cancer cells in vitro. The MCF-7 cells were exposed to both PDOX and DOX, and cytotoxicities, cell cycle and P53/P21 signaling alterations were studied. Abundant cathepsin B was found in the MCF-7 cells, and treatment with PDOX and DOX triggered dose- and time-dependent cytotoxicity and resulted in a significant reduction in cell viability. The IC50 of PDOX and DOX was 3.91 and 0.94 µmol/L, respectively. Both PDOX and DOX caused an up-regulation of the P53/P21-related signal pathway, and PDOX significantly increased expression of P53 and caspase 3, and arrested the cell cycle at the G1/G2 phase. As compared with DOX, PDOX reduced toxicities, and it may have different action mechanisms on breast cancer cells.

Targeting therapy of hepatocellular carcinoma with doxorubicin prodrug PDOX increases anti-metastatic effect and reduces toxicity: a preclinical study.

BACKGROUND: This study was to investigate the effects and safety of cathepsin B-cleavable doxorubicin (DOX)-prodrug (PDOX) for targeting therapy of metastatic human hepatocellular carcinoma (HCC) using DOX as a positive control drug. METHODS: The orthotopic nude mice model of highly metastatic HCC was established and the animals were randomized and treated with PDOX, DOX and saline, respectively. Hematology, biochemistry and tumor markers were studied. At autopsy, liver tumor weight and size, ascites, abdominal lymph nodes metastases, experimental peritoneal carcinomatosis index (ePCI), and tumor-host body weight ratio were investigated. Immunohistochemical studies and western blotting were done to investigate key molecules involved in the mechanism of action. RESULTS: Compared with Control, both PDOX and DOX could similarly and significantly reduce liver tumor weight and tumor volume by over 40%, ePCI values, retroperitoneal lymph node metastases and lung metastases and serum AFP levels (P < 0.05). The PDOX group had significantly higher WBC than the DOX group (P < 0.05), and higher PLT than Control (P < 0.05). Serum BUN and Cr levels were lower in the PDOX group than DOX and Control groups (P < 0.05). Compared with Control, DOX increased CK and CK-MB; while PDOX decreased CK compared with DOX (P < 0.05). Multiple spotty degenerative changes of the myocardium were observed in DOX-treated mice, but not in the Control and PDOX groups. PDOX could significantly reduce the Ki-67 positive rate of tumor cells, compared with DOX and Control groups. PDOX produced the effects at least via the ERK pathway. CONCLUSION: Compared with DOX, PDOX may have better anti-metastatic efficacy and reduced side effects especially cardio-toxicities in this HCC model.

Cathepsin B cleavable novel prodrug Ac-Phe-Lys-PABC-ADM enhances efficacy at reduced toxicity in treating gastric cancer peritoneal carcinomatosis: an experimental study.

BACKGROUND: Doxorubicin (Adriamycin) is effective in gastric cancer treatment, but with severe dose-dependent toxicities. A novel prodrug of doxorubicin (Ac-Phe-Lys-PABC-ADM) is designed to deliver free doxorubicin relying on cathepsin B and reduce side effects. The authors examined the antitumor effect and toxicities of Ac-Phe-Lys-PABC-ADM against gastric cancer peritoneal carcinomatosis. METHODS: SGC-7901 gastric cancer cell line was used for the study. The in vitro study investigated the effects of doxorubicin and Ac-Phe-Lys-PABC-ADM on cell growth dynamics and cell cycle. The in vivo study investigated the efficacy and toxicity of Ac-Phe-Lys-PABC-ADM on a nude mice model of peritoneal carcinomatosis, with doxorubicin as positive control. RESULTS: In the in vitro study, Ac-Phe-Lys-PABC-ADM had a lower dose-dependent inhibitory effect on SGC-7901 cells. In the in vivo study of control, doxorubicin, and Ac-Phe-Lys-PABC-ADM groups, the median experimental peritoneal carcinomatosis indexes were 6, 1.5, and 1, respectively (P = .004); the body weights were 24.32 ± 1.40 g, 18.40 ± 2.97 g, and 23.61 ± 0.80 g, respectively (P = .000). Biochemical studies showed that Ac-Phe-Lys-PABC-ADM had significantly lower toxicities on the bone marrow, liver, kidney, and particularly heart. Histopathological studies of the control, doxorubicin, and Ac-Phe-Lys-PABC-ADM groups found significant myocardium toxicities in 3, 7, and 4 animals, respectively. CONCLUSIONS: Ac-Phe-Lys-PABC-ADM could be an effective molecular targeting drug to treat gastric cancer peritoneal carcinomatosis with enhanced efficacy and reduced toxicity.

Experimental evidence of good efficacy and reduced toxicity with peptide-doxorubicin to treat gastric cancer.

BACKGROUND: To compare the efficacy and toxicity of peptide-doxorubicin (PDOX) and doxorubicin (DOX) on nude mice models of human gastric cancer. RESULTS: Both PDOX and DOX could significantly inhibit tumor growth compared with Control (P < 0.05) in both subcutaneous and orthotopic models. Animal survival was much better in PDOX group than DOX group. In peripheral blood test, PDOX group had significantly higher levels of platelets than the Control (P < 0.05), and lymphocyte lower than Control (P < 0.05). There were no significant differences on liver, kidney and cardiac function parameters among three groups (P > 0.05). Immunohistochemistry showed that treatment groups had much higher Tunel than Control (P < 0.05), and PDOX had significantly lower Ki-67 than doxorubicin and Control group (P < 0.01). Western blotting showed that PDOX caused much higher expressions of P53, P21, Aparf-1, pro- and cleaved-caspase 3, compared with DOX. CONCLUSION: Compared with DOX, PDOX has increased effects but much decreased toxicity in treating animal model of gastric cancer. MATERIALS AND METHODS: Animals in subcutaneous model were randomized into Control, doxorubicin, PDOX-L, PDOX-M, and PDOX-H groups. Animals in surgical orthotopic implantation model were randomized into Control, doxorubicin and, peptide-doxorubicin groups. The animals were treated, monitored and examined following a set protocol.

Mechanism-Based Enzyme Inactivation Using an Allyl Sulfoxide-Allyl Sulfenate Ester Rearrangement1.

2-Amino-4-chloro-5-(p-nitrophenylsulfinyl)pentanoic acid (1) has been synthesized and shown to induce mechanism-based inactivation of two pyridoxal phosphate dependent enzymes: (1) cystathionine γ-synthetase, which catalyzes a γ-replacement reaction in bacterial methionine biosynthesis; and (2) methionine γ-lyase, which catalyzes a γ-elimination reaction in bacterial methionine breakdown. The inactivations are irreversible and display saturation kinetics. Each enzyme incorporates roughly 1 mol of tritium per mol of enzyme monomer when inactivated by 2-amino-4-chloro-5-(p-nitro[3H]phenyl-sulfinyl)pentanoic acid (la), confirming that the modification of each protein is covalent and stoichiometric. Substoichiometric labeling (0.12 mol of tritium per mol of enzyme monomer) is given when methionine γ-lyase is fully inactivated by 2-amino-4-chloro-5-[3H]-5-p-nitrophenylsulfinyl)pentanoic acid (lb). Both enzymes, inactivated by 1, are susceptible to reactivation by thiols. Inactivated cystathionine γ-synthetase recovers 25% of its catalytic activity upon incubation with excess dithiothreitol, while methinonine γ-lyase is 100% reactivated by dithiothreitol, mercaptoethanol, and mercaptopropionate. Reactivation generates p-nitrophenylthiolate anion, which forms, in the case of methionine γ-lyase, stoichiometrically with enzyme reactivated. Both enzymes are "protected" from inactivation by 1 in the presence of thiols, which simultaneously generates p-nitrophenylthiol. In the presence of dithiothreitol, the protection reaction gives p-nitrophenylthiol production with pseudo-first-order kinetics. 2-Amino-4-chloro-5-(p-tolylsulfinyl)pentanoic acid (2) and 2-amino-4-(p-nitrophenylsulfinyl)-5-chloropentanoic acid (3), the reverse regioisomer of 1, have also been prepared and give no evidence of inactivation of either enzyme. The data are taken to indicate a novel form of suicide inactivation (Scheme II) wherein ß-carbanion-assisted γ-halide elimination generates an allyl sulfoxide-enzyme-pyridoxal adduct (4) which undergoes spontaneous 2,3-sigmatropic rearrangement to an electrophilic allyl sulfenate ester (5). The latter is then captured by an enzymic nucleophile to give an inactive enzyme 6, which may be a mixed disulfide or, less likely, a sulfenamide.

Monoclonal antibody conjugates of doxorubicin prepared with branched peptide linkers: inhibition of aggregation by methoxytriethyleneglycol chains.

High mole ratio BR96 immunoconjugates were synthesized using branched peptide-doxorubicin linkers designed to liberate doxorubicin following antigen-specific internalization into lysosomes. However, these immunoconjugates are highly prone to noncovalent, dimeric aggregation. We hypothesize that this is due to (1) the hydrophobic nature of the peptides, (2) the loss of positive charge upon amide formation at the 3'-amino group of doxorubicin, and (3) the proximity of the peptide hydrophobic residues to form efficient intermolecular stacking interactions. By introducing a hydrophilic methoxytriethylene glycol chain onto the doxorubicin portion of the branched peptide linkers, aggregation has been eliminated or greatly reduced in the immunoconjugate products. The methoxytriethylene glycol chain was linked to the doxorubicin moiety of the linker via a hydrazone bond that is stable at pH 7 but hydrolyzes rapidly at pH 5 to release free drug. BR96 immunoconjugates synthesized from methoxytriethylene glycol-modified branched peptide-doxorubicin linkers are highly potent and immunospecific in vitro. The data suggest that the methoxytriethylene glycol chain hydrolyzes as designed upon antigen-specific internalization into tumor lysosomes in vitro, where enzymatic degradation of the peptide linker releases free doxorubicin.

Amines That Transport Protons across Bilayer Membranes: Synthesis, Lysosomal Neutralization, and Two-Phase pK(a) Values by NMR.

It is desirable to be able to control the pH of lysosomes. A collection of lipophilic, nitrogenous bases, designed to act as membrane-active, catalytic proton transfer agents, were prepared and their effective pK(a)s measured in a vigorously stirred, two-phase system. One phase was a phosphate buffer whose pH was varied over the range ca. 1-11. The other was an immiscible, deuterated organic solvent in which the compounds preferentially resided even when protonated. When chemical shift changes versus the pH of the buffer were plotted, clear pK(a) curves were generated that are relevant to transmembrane proton transfer behavior. The two-phase pK(a)s increased with increasing counterion lipophilicity and with increasing organic solvent polarity. The compounds were also tested for their ability to neutralize the acidity of lysosomes, a model for other acidic vesicles involved in drug sorting. The most successful of these, imidazole 6a, has >100 times the neutralizing power of ammonia, a standard lysosomotropic amine, causing a 1.7 unit rise in lysosomal pH of RAW cells at 0.1 mM, compared to a 0.2 and 1.4 unit rise for ammonium chloride at 0.1 and 10 mM, respectively.

Anticancer effects of Ac­Phe­Lys­PABC­doxorubicin via mitochondria­centered apoptosis involving reactive oxidative stress and the ERK1/2 signaling pathway in MGC­803 cells.

Ac­Phe­Lys­PABC­DOX (PDOX) is a smart doxorubicin (DOX) prodrug designed to decrease toxicities while maintaining the potent anticancer effects of DOX. The present study aimed to elucidate the molecular mechanisms of action of PDOX using MGC­803 gastric cancer cells as a model. The cells were treated with both PDOX and DOX, and cytotoxicities, cell cycle analysis, reactive oxygen species (ROS) generation, mitochondrial damage and ERK1/2 signaling pathway alterations were studied. Abundant cathepsin B expression was observed in the MGC­803 cells, and treatment with PDOX and DOX triggered dose­dependent cytotoxicity and resulted in a significant reduction in cell viability. IC50 of PDOX and DOX was 14.9 and 4.9 µM, respectively. Both PDOX and DOX significantly decreased p­ERK1/2, increased ROS generation, reduced mitochondrial membrane potential, caused mitochondrial swelling and arrested the cell cycle at the G2/S phase, and these effects were more pronounced for PDOX than for DOX. PDOX and DOX have different mechanisms of action, particularly the mitochondria­centered intrinsic apoptosis involving reactive oxidative stress and the ERK1/2 signaling pathway.

Synthesis of an immunoconjugate of camptothecin.

The first immunoconjugate of camptothecin has been synthesized wherein the drug is attached to the tumor-recognizing antibody BR96 via a Cathepsin B cleavable linker. Endocytosis of the immunoconjugate upon binding to the tumor cell followed by enzymatic cleavage of the linker inside the endosome ensures tumor-specific release of the drug. In this way, it is hoped that the dose-limiting side effects associated with camptothecin can be eliminated while the antitumor activity is preserved.

BR96 conjugates of highly potent anthracyclines.

The 6-maleimidocaproylhydrazone derivatives of highly potent antitumor agents 5-Diacetoxypentyldoxorubicin and Morpholinodoxorubicin were synthesized and conjugated to monoclonal antibody BR96 and control IgG. Immunoconjugate molar ratios were generally 7.5-8.5, and dimer aggregate levels were low. The linkers released parent drug at lysosomal pH 5, while they remained stable at neutral pH. BR96 conjugates were highly potent and antigen specific in vitro. The BR96-DAPDOX conjugate demonstrated an IC(50) of 0.03 micrometer and was at least 300-fold more potent than a non-binding IgG-DAPDOX control conjugate.

Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity.

The anticancer drug doxorubicin (DOX) has been linked to chimeric BR96, an internalizing monoclonal antibody that binds to a Lewis(y)-related, tumor-associated antigen, through two lysosomally cleavable dipeptides, Phe-Lys and Val-Cit, giving immunoconjugates 72 and 73. A self-immolative p-aminobenzyloxycarbonyl (PABC) spacer between the dipeptides and the DOX was required for rapid and quantitative generation of free drug. DOX release from model substrate Z-Phe-Lys-PABC-DOX 49 was 30-fold faster than from Z-Val-Cit-PABC-DOX 42 with the cysteine protease cathepsin B alone, but rates were identical in a rat liver lysosomal preparation suggesting the participation of more than one enzyme. Conjugates 72 and 73 showed rapid and near quantitative drug release with cathepsin B and in a lysosomal preparation, while demonstrating excellent stability in human plasma. Against tumor cell lines with varying levels of BR96 expression, both conjugates showed potent, antigen-specific cytotoxic activity, suggesting that they will be effective in delivering DOX selectively to antigen-expressing carcinomas.

Doxorubicin immunoconjugates containing bivalent, lysosomally-cleavable dipeptide linkages.

Bivalent doxorubicin (DOX)-dipeptides (16a-c) were prepared and conjugated to the monoclonal antibody BR96. The dipeptides are cleaved by lysosomal proteases following internalization of the resulting immunoconjugates. Conjugate 18b demonstrated antigen-specific in vitro tumor cell killing activity (IC(50)=0.2 microM) that was equipotent to DOX with a near doubling of drug molecules/MAb. Size exclusion chromatography showed 18b to be a noncovalent dimer that was formed immediately upon conjugation.

The design of potent hydrazones and disulfides as cathepsin S inhibitors.

The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis. The nature and mechanism of these adducts was explored. Kinetic analysis of the benzoyl hydrazones indicate that these inhibitors are acting as irreversible inhibitors of Cathepsin S. Additionally, the benzoylhydrazones were shown to be potent inhibitors of Cathepsin S processing of Class II associated invariant peptide both in vitro and in vivo.