Potent antitumor N-mustard derivatives of 9-anilinoacridine, synthesis and antitumor evaluation

A series of 9-anilinoacridine N-mustard derivatives, in which the alkylating N-mustard residue was linked to the C-3' or C-4' position of the anilino ring with an O-ethylene spacer, was synthesized and evaluated for cytotoxicity against human lymphoblastic leukemic cells (CCRF-CEM) in culture. The results showed that all of the new compounds exhibited potent cytotoxicity with IC(50) values ranging from 0.002 to 0.7 microM, which were as potent or significantly more potent than 3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA). Compound 9 did not exhibit cross-resistance against both vinblastine-resistant (CCRF-CEM/VBL) and taxol-resistant (CCRF-CEM/taxol) cells. Additionally, compound 9 demonstrated potent antitumor effect in nude mice bearing human breast carcinoma MX-1 xenografts, resulting in complete tumor remission in two out of three mice at the maximal dose of 1-2mg/kg (Q3Dx7) or 3mg/kg (Q4Dx5) via intravenous injection.     

Potent antitumor 9-anilinoacridines bearing an alkylating N-mustard residue on the anilino ring: synthesis and biological activity

A series of N-mustard derivatives of 9-anilinoacridine was synthesized for antitumor and structure-activity relationship studies. The alkylating N-mustard residue was linked to the C-3' or C-4' position of the anilino ring with an O-ethylene (O-C(2)), O-butylene (O-C(4)), and methylene (C(1)) spacer. All of the new N-mustard derivatives exhibited significant cytotoxicity in inhibiting human lymphoblastic leukemic cells (CCRF-CEM) in culture. Of these agents, (3-(acridin-9-ylamino)-5-{2-[bis (2-chloroethyl)amino]ethoxy}phenyl)methanol (10) was subjected to antitumor studies, resulting in an approximately 100-fold more potent effect than its parent analogue 3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA) in inhibiting the growth of human lymphoblastic leukemic cells (CCRF-CEM) in vitro. This agent did not exhibit cross-resistance against vinblastine-resistant (CCRF-CEM/VBL) or Taxol-resistant (CCRF-CEM/Taxol) cells. Remarkably, the therapeutic effect of 10 at a dose as low as one tenth of the Taxol therapeutic dose [i.e., 1-2mg/kg (Q3Dx7) or 3mg/kg (Q4Dx5); intravenous injection] on nude mice bearing human breast carcinoma MX-1 xenografts resulted in complete tumor remission in two out of three mice. Furthermore, 10 yielded xenograft tumor suppression of 81-96% using human T-cell acute lymphoblastic leukemia CCRF-CEM, colon carcinoma HCT-116, and ovarian adenocarcinoma SK-OV-3 tumor models.     

Design, synthesis and antitumor evaluation of phenyl N-mustard-quinazoline conjugates

A series of N-mustard-quinazoline conjugates was synthesized and subjected to antitumor studies. The N-mustard pharmacophore was attached at the C-6 of the 4-anilinoquinazolines via a urea linker. To study the structure-activity relationships of these conjugates, various substituents were introduced to the C-4 anilino moiety. The preliminary antitumor studies revealed that these agents exhibited significant antitumor activity in inhibiting various human tumor cell growths in vitro. Compounds 21b, 21g, and 21h were selected for further antitumor activity evaluation against human breast carcinoma MX-1 and prostate PC-3 xenograft in animal model. These agents showed 54-75% tumor suppression with low toxicity (5-7% body-weight changes). We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay and inhibited cell cycle arrest at G2/M phase.     

A Multifunctional Drug Combination Shows Highly Potent Therapeutic Efficacy against Human Cancer Xenografts in Athymic Mice

The tumor microenvironment plays a crucial role during tumor development. Integrated combination of drugs that target tumor microenvironment is a promising approach to anticancer therapy. Here, we report a multifunctional combination of low-cytotoxic drugs composed of dipyridamole, bestatin and dexamethasone (DBDx) which mainly acts on the tumor microenvironment shows highly potent antitumor efficacy in vivo. In mouse hepatoma H22 model, the triple drug combination showed synergistic and highly potent antitumor efficacy. The combination indices of various combinations of the triple drugs were between 0.2 and 0.5. DBDx inhibited the growth of a panel of human tumor xenografts and showed no obvious systemic toxicity. At tolerated doses, DBDx suppressed the growth of human hepatocellular carcinoma BEL-7402, HepG2, and lung adenocarcinoma A549 xenografts by 94.5%, 93.7% and 96.9%, respectively. Clonogenic assay demonstrated that DBDx showed weak cytotoxicity. Western blot showed that Flk1 and Nos3 were down-regulated in the DBDx-treated group. Proteomic analysis showed that DBDx mainly affected the metabolic process and immune system process; in addition, the angiogenesis and VEGF signaling pathway were also affected. Conclusively, DBDx, a multifunctional drug combination of three low-cytotoxic drugs, shows synergistic and highly potent antitumor efficacy evidently mediated by the modulation of tumor microenvironment. Based on its low-cytotoxic attributes and its broad-spectrum antitumor therapeutic efficacy, this multifunctional combination might be useful in the treatment of cancers, especially those refractory to conventional chemotherapeutics.     

Design, synthesis, and biological evaluation of novel water-soluble N-mustards as potential anticancer agents

A series of novel water-soluble N-mustard-benzene conjugates bearing a urea linker were synthesized. The benzene moiety contains various hydrophilic side chains are linked to the meta- or para-position of the urea linker via a carboxamide or an ether linkage. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and therapeutic efficacy against human tumor xenografts in vivo. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft and significant suppression against prostate adenocarcinoma PC3 xenograft were achieved by treating with compound 9aa′ at the maximum tolerable dose with relatively low toxicity. We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay. A pharmacokinetic profile of the representative 9aa′ in rats was also investigated. The current studies suggest that this agent is a promising candidate for preclinical studies.     

Antitumor effects, cell selectivity and structure-activity relationship of a novel antimicrobial peptide polybia-MPI

A novel antimicrobial peptide, polybia-MPI, was purified from the venom of the social wasp Polybia paulista. It has potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, but causing no hemolysis to rat erythrocytes. To date, there is no report about its antitumor effects on any tumor cell lines. In this study we synthesized polybia-MPI and studied its antitumor efficacy and cell selectivity. Our results revealed that polybia-MPI exerts cytotoxic and antiproliferative efficacy by pore formation. It can selectively inhibit the proliferation of prostate and bladder cancer cells, but has lower cytotoxicity to normal murine fibroblasts. In addition, to investigate the structure–activity relationship of polybia-MPI, three analogs in which Leu⁷, Ala⁸ or Asp⁹ replaced by l-Pro were designed and synthesized. l-Pro substitution of Leu⁷ or Asp⁹ significantly reduces the content of -helix conformation, and l-Pro substitution of Ala⁸ can disrupt the -helix conformation thoroughly. The l-Pro substitution induces a significant reduction of antitumor activity, indicating that the -helix conformation of polybia-MPI is important for its antitumor activity. In summary, polybia-MPI may offer a novel therapeutic strategy in the treatment of prostate cancer and bladder cancer, considering its relatively lower cytoxicity to normal cells.     

CD40 ligand promotes priming of fully potent antitumor CD4(+) T cells in draining lymph nodes in the presence of apoptotic tumor cells.

The presence or absence of CD4(+) T cell help can determine the direction of adaptive immune responses toward either cross-priming or cross-tolerance. It has been demonstrated that interactions of CD40-CD40 ligand can replace CD4(+) T cell help and enable dendritic cells to prime cytotoxic T cells. Here, we demonstrate that antitumor reactivity induced in regional lymph nodes (LNs) by s.c. injection of CD40 ligand (CD40L)-transduced tumor (MCA205 CD40L) showed far superior therapeutic efficacy against established brain tumors of a weakly immunogenic fibrosarcoma, MCA205, when adoptively transferred. Coinjection of apoptotic, but not necrotic parental tumor cells with CD40L-expressing tumor cells caused a strong synergistic induction of antitumor reactivity in tumor-draining LNs. Freshly isolated T cells from LNs immunized with apoptotic parental tumor cells and MCA205 CD40L were capable of mediating regression of the parental tumor in vivo. In contrast, T cells derived from LNs immunized without MCA205 CD40L required ex vivo anti-CD3/IL-2 activation to elicit therapeutic activity. On anti-CD3/IL-2 activation, cells from LNs immunized with MCA205 CD40L exhibited superior per cell antitumor reactivity. An in vitro depletion study revealed that either CD4(+) or CD8(+) T cells could mediate therapeutic efficacy but that the antitumor efficacy mediated by CD4(+) T cells was far superior. Cytosolic flow cytometric analyses indicated that priming of CD4(+) cells in LNs draining CD40L-expressing tumors was polarized to the Th1 type. This is the first report that fully potent antitumor CD4(+) T cell priming was promoted by s.c. injection of CD40L-transduced tumor in the presence of apoptotic tumor cells.     

Novel DNA-directed alkylating agents: Design,synthesis and potent antitumor effect of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker

A series of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker was synthesized for antitumor evaluation. The carbamate or carbonate linker is able to lower the reactivity of the phenyl N-mustard pharmacophore and thus, these conjugates are rather chemically stable. The in vitro studies revealed that these derivatives possessed significant cytotoxicity with IC₅₀ in sub-micromolar range in inhibiting human lymphoblastic leukemia (CCRF-CEM), breast carcinoma (MX-1), colon carcinoma (HCT-116) and human non-small cell lung cancer (H1299) cell growth in vitro. Compounds 10a, 10b, 10e, 10i, and 15a were selected for evaluating their antitumor activity in nude mice bearing MX-1 and HCT-116 xenografts. Remarkably, total tumor remission was achieved by these agents with only one cycle of treatment. Interestingly, no tumor relapse was found in mice treated with 10a over 129 days. This agent is capable of inducing DNA interstrand cross-linking in human non-small lung cancer H1299 cells in a dose dependent manner by modified comet assay and has a long half-life in rat plasma.     

Design,synthesis and evaluation of acridine derivatives as multi-target Src and MEK kinase inhibitors for anti-tumor treatment

Clinical studies have shown enhanced anticancer effects of combined inhibition of Src and MEK kinases. Development of multi-target drugs against Src and MEK is of potential therapeutic advantage against cancers. As a follow-up of our previous studies, and by using molecular docking method, we designed and synthesized a new series of 9-anilinoacridines containing phenyl-urea moieties as potential novel dual Src and MEK inhibitors. The anti-proliferative assays against K562 and HepG-2 tumor cells showed that most of the derivatives displayed good cytotoxicity in vitro. In particular, kinase inhibition assays showed that compound 8m inhibited Src (59.67%) and MEK (43.23%) at 10 μM, and displayed moderate inhibitory activity against ERK and AKT, the downstream effectors of both Src and MEK. Moreover, compound 8m was found to induce K562 cells apoptosis. Structure–activity relationships of these derivatives were analyzed. Our study suggested that acridine scaffold, particularly compound 8m, is of potential interest for developing novel multi-target Src and MEK kinase inhibitors.     

Marine lysozyme from a marine bacterium that inhibits angiogenesis and tumor growth

Recent studies suggest that lysozyme, rich in hen egg, has an antitumor function. In the present study, we investigated the antitumor and antiangiogenesis effects of a newly isolated marine lysozyme both in vitro and in vivo. First, we showed that this marine-derived lysozyme specifically inhibits the proliferation of endothelial cells (ECV304) in a dose-dependent manner with no cytotoxicity (IC₅₀ = 3.64 μM). Second, we showed that this marine lysozyme directly suppresses neovascularization in chicken embryos using chorioallantoic membrane assay. Third, we demonstrated that this marine lysozyme markedly inhibits tumor growth in mice bearing either sarcoma 180 or hepatoma 22. Unexpectedly, hen egg lysozyme has no effects on the proliferation of endothelial cells in vitro or neovascularization in chicken embryos or tumor growth in nude mice at the same dosage range. Taken together, our studies clearly show that the newly identified marine lysozyme is a potent antitumor molecule, which may inhibit tumor growth and inhibit angiogenesis. We believe that this marine lysozyme may have a therapeutic value in antitumor drug development.     

Structure-based design,synthesis and biological testing of etoposide analog epipodophyllotoxin–N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA

Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series of etoposide analog epipodophyllotoxin–N-mustard hybrid compounds were designed, synthesized and biologically characterized. These hybrids were designed to alkylate nucleophilic protein residues on topoisomerase II and thus produce inactive covalent adducts and to also alkylate DNA. The most potent hybrid had a mean GI₅₀ in the NCI-60 cell screen 17-fold lower than etoposide. Using a variety of in vitro and cell-based assays all of the hybrids tested were shown to target topoisomerase II. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles matching both etoposide and the N-mustard compounds from which they were derived. These results supported the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA.     

A novel anti-Cyr61 antibody inhibits breast cancer growth and metastasis in vivo

Cysteine-rich protein 61(CCN1/Cyr61) has been implicated as an important mediator in proliferation and metastasis of breast cancer, which indicated that blockage of Cyr61 might be a potent target for breast cancer treatment. However, the antitumor effect of anti-Cyr61 antibodies on breast cancer in vivo has not been reported so far. In this study, we reported the effect and likely mechanism of generated anti-human Cyr61 monoclonal antibodies (mAb) on Cyr61 high expression line MDA-MB-231, known as a highly malignant and invasive human breast cancer cell line, at aspects of proliferation and migration in vitro and in vivo. We found the mAb, denoted as 093G9, revealed inhibitory effects on MDA-MB-231 cell proliferation, migration, and invasion through downregulation of both AKT and ERK phosphorylation in vitro compared with its isotype control. 093G9 also showed significant efficacy on suppressing primary tumor growth and spontaneous lymph node metastasis in in vivo mouse model. The specific epitope recognized by 093G9 was identified to be ¹⁴⁰LPNLGCP¹⁴⁶, adjacent to the VWC domain of Cyr61 by Ph.D.-C7C phage library display system. Our study provides direct evidence that Cyr61 can be a potent therapeutic target for patients who bear high Cyr61 expression breast cancer. Furthermore, the mAb, 093G9 developed in our laboratory, has shown a promising therapeutic characteristic in breast cancer.     

Dichloroacetate (DCA) enhances tumor cell death in combination with oncolytic adenovirus armed with MDA-7/IL-24

Dichloroacetate (DCA) is a metabolic modulator for the treatment of lactic acidosis and inherited mitochondrial diseases. A recent study showed that DCA treatment could induce apoptosis in many kinds of tumor cell lines via mitochondrial apoptotic pathway while sparing normal cells. ONYX-015 (dl 1520) is one of the oncolytic adenoviruses developed by the deletion of E1B-55kD gene of type 5 adenoviral DNA, and it replicates efficiently and selectively in tumor cells. ZD55-IL-24, an E1B-55kD deleted oncolytic adenovirus carrying interleukin-24 (IL-24, also called melanoma differentiation associated gene-7), had showed potent antitumor efficacy in a variety of tumor cells and exerted no apparent toxicity on normal cells. Given both the good therapeutic effect and low toxicity of these agents, here we investigated whether DCA in combination with ZD55-IL-24 or ONYX-015 could have more efficient antitumor activity in vitro experiments. Therefore, we tested the cytotoxicity of combination therapy in normal hepatic cells L-02 and QSG-7701 using the MTT assay. Our results showed that DCA combined with ONYX-015 or ZD55-IL-24 exhibited more potent antitumor activity than DCA or virus alone, and the combination treatment did not have superimposed toxicities in normal cells. Thus, a novel combination therapy associating oncolytic adenoviruses with relatively low toxic drug without severe side effects was proposed.     

Synthesis and antitumor activity of 5-(9-acridinylamino)anisidine derivatives

A series of 5-(9-acridinylamino)anisidines were synthesized by condensing methoxy-substituted 1,3-phenylenediamines (10 and 11) with 9-chloroacridine derivatives to form 5-(9-acridinylamino)-m-anisidines (AMAs, 14a-e) and 5-(9-acridinylamino)-o-anisidines (AOAs, 15a-e). 5-(9-Acridinylamino)-p-anisidines (APAs, 17a-e) were synthesized by reacting 2-methoxy-5-nitroaniline (12) with 9-anilinoacridines, followed by reduction. The cytotoxic inhibition of growth of various human tumor cells in culture, inhibitory effects against topoisomerase II, and DNA interaction of these agents were studied. The structure-activity relationship studies revealed the following degree of potency: AOAs > AMAs > APAs. They also revealed that the newly synthesized derivatives bearing CONH(2)NH(2)NMe(2) and Me substituents at C4 and C5 positions of the acridine chromophore (i.e., AMA 14e, AOA 15e, and APA 17e) exhibited significant cytotoxicity against human tumor cell growth in vitro. AOA (15e) was the most potent among these derivatives, which resulted in 60% suppression of tumor volume at a dose of 20 mg/kg (Q2D x 9), intravenous injection on day 26 in nude mice bearing human breast carcinoma MX-1 xenografts.     

Successful adoptive immunotherapy of murine poorly immunogenic tumor with specific effector cells generated from gene-modified tumor-primed lymph node cells

We previously reported that cytokine gene transfer into weakly immunogenic tumor cells could enhance the generation of precursor cells of tumor-reactive T cells and subsequently augment antitumor efficacy of adoptive immunotherapy. We investigated whether such potent antitumor effector T cells could be generated from mice bearing poorly immunogenic tumors. In contrast to similarly modified weakly immunogenic tumors, MCA102 cells, which are chemically induced poorly immunogenic fibrosarcoma cells transfected with cDNA for IL-2, IL-4, IL-6, IFN-gamma, failed to augment the host immune reaction. Because priming of antitumor effector T cells in vivo requires two important signals provided by tumor-associated Ags and costimulatory molecules, these tumor cells were cotransfected with a B7-1 cDNA. Transfection of both IFN-gamma and B7-1 (MCA102/B7-1/IFN-gamma) resulted in regression of s.c. tumors, while tumor transfected with other combinations of cytokine and B7-1 showed progressive growth. Cotransfection of IFN-gamma and B7-1 into other poorly immunogenic tumor B16 and LLC cells also resulted in the regression of s.c. tumors. Cells derived from lymph nodes draining MCA102/B7-1/IFN-gamma tumors showed potent antitumor efficacy, eradicating established pulmonary metastases, but this effect was not seen with parental tumors. This mechanism of enhanced antitumor efficacy was further investigated, and T cells with down-regulated L-selectin expression, which constituted all the in vivo antitumor reactivity, were significantly increased in lymph nodes draining MCA102/B7-1/IFN-gamma tumors. These T cells developed into potent antitumor effector cells after in vitro activation with anti-CD3/IL-2. The strategy presented here may provide a basis for developing potent immunotherapy for human cancers.     

Synthesis and antitumor activity of 4-hydroxycoumarin derivatives

A series of 4-hydroxycoumarin derivatives was prepared and evaluated for antitumor activity. The key fragments were 2a-c, 5c, 12b, 13b, 17, and 18 which were prepared via dianion ring cyclization, Friedel-Crafts acylation, and Reformatsky reaction. Compound 20b showed the most potent antitumor activity among the total 12 derivatives and compounds 19a and 19b exhibited efficacy comparable to etoposide in vitro antitumor activity.     

New insights into IL-12-mediated tumor suppression

During the past two decades, interleukin-12 (IL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models. Through pleiotropic effects on different immune cells that form the tumor microenvironment, IL-12 establishes a link between innate and adaptive immunity that involves different immune effector cells and cytokines depending on the type of tumor or the affected tissue. The robust antitumor response exerted by IL-12, however, has not yet been successfully translated into the clinics. The majority of clinical trials involving treatment with IL-12 failed to show sustained antitumor responses and were associated to toxic side effects. Here we discuss the therapeutic effects of IL-12 from preclinical to clinical studies, and will highlight promising strategies to take advantage of the antitumor activity of IL-12 while limiting adverse effects.     

Combined immunization with adjuvant molecules poly(I:C) and anti-CD40 plus a tumor antigen has potent prophylactic and therapeutic antitumor effects

The low immunogenicity of malignant cells is one of the causes responsible for the lack of antitumor immune responses. Thus, development of new therapeutic strategies aimed at enhancing presentation of tumor antigens to T cells is a main goal of cancer immunotherapy. With this aim, we studied the efficacy of administering adjuvants poly(I:C) and agonistic anti-CD40 antibody plus a tumor antigen. Joint intravenous immunization with these adjuvants and a model tumor antigen (ovalbumin) was able to synergistically induce potent and long lasting antitumor T-cell responses. These responses protected against challenge with E.G7–OVA tumor cells in prophylactic short- and long-term vaccination. In a therapeutic setting, repeated intratumor administration of adjuvants plus antigen was able to reject established tumors in all treated animals, leading in some cases to the rejection of both locally treated and untreated tumors. Antitumor immune responses induced by these protocols were mediated not only by T-cells but also by NK cells. In conclusion, combined administration of adjuvants poly(I:C) and anti-CD40 plus a tumor antigen is an efficient strategy for prophylactic and therapeutic antitumor vaccination.     

Antitumor therapeutic effects of e7 subunit and DNA vaccines in an animal cervical cancer model: antitumor efficacy of e7 therapeutic vaccines is dependent on tumor sizes, vaccine doses, and vaccine delivery routes

We previously reported that E7 subunit and DNA vaccines are both capable of inducing antitumor protection through induction of antigen-specific CTL. In this study, we investigated their ability to control established tumors according to tumor size, vaccine doses, and vaccine delivery routes. Antitumor therapeutic efficacy of both vaccine types was dependent on tumor burden. However, E7 subunit vaccines induced a higher level of antitumor therapeutic activities at the tested dose compared to DNA vaccines. This was concomitant with induction of antibody, CTL, and IFN-gamma responses, as well as histologic changes (heavy infiltration of lymphocytes and presence of apoptotic bodies). In vaccine dose titration assays, 50 and 100 microg of DNA vaccines exhibited an equivalent antitumor efficacy to 0.5 and 1 microg of E7 subunit vaccines, respectively, i.e., a 100-fold difference in E7 dosage, suggesting the importance of vaccine doses for achieving antitumor immunity. Furthermore, tumors of a larger size were controlled by intratumoral injection with E7 subunit vaccines, underscoring the importance of vaccine delivery routes for antitumor therapeutic efficacy. Thus, these data suggest that antitumor therapeutic efficacy of E7 therapeutic vaccines is determined by vaccine doses, vaccine delivery routes, and tumor sizes, and that these vaccines could be another addition to conventional therapy modalities against cervical cancer.     

Efficacy and toxicity elicited by recombinant interferons alpha and gamma when administered in combination to tumor-bearing mice

Administration of rHuIFN-alpha A/D and rMuIFN-gamma as single agents to tumor-bearing mice resulted in a dose-related antitumor effect in each of the six models studied. When the IFNs were given in combination, the effects varied between the tumor systems. No increase in efficacy was seen in mice bearing B16-F10 melanoma or M5076 reticulum cell sarcoma while additive antitumor activity was shown in the KA31 fibrosarcoma and P388 leukemia systems. Mice inoculated with L1210 lymphoma or colon 38 carcinoma, however, revealed enhanced efficacy which was greater than additive. The data also reveal that combination of IFNs alpha and gamma administered to normal and tumor-bearing mice resulted in toxicity which was not predicted by the appropriate doses of the single agents. These studies suggest that combination of IFNs alpha and gamma may provide greater therapeutic utility than the single agents and underscore the need for additional, carefully designed preclinical and clinical efforts.