Acid ceramidase expression modulates the sensitivity of A375 melanoma cells to dacarbazine

Dacarbazine (DTIC) is the treatment of choice for metastatic melanoma, but its response in patients remains very poor. Ceramide has been shown to be a death effector and to play an important role in regulating cancer cell growth upon chemotherapy. Among ceramidases, the enzymes that catabolize ceramide, acid ceramidase (aCDase) has been implicated in cancer progression. Here we show that DTIC elicits a time- and dose-dependent decrease of aCDase activity and an increase of intracellular ceramide levels in human A375 melanoma cells. The loss of enzyme activity occurred as a consequence of reactive oxygen species-dependent activation of cathepsin B-mediated degradation of aCDase. These events preceded autophagic features and loss of cell viability. Down-regulation of acid but not neutral or alkaline ceramidase 2 resulted in elevated levels of ceramide and sensitization to the toxic effects of DTIC. Conversely, inducible overexpression of acid but not neutral ceramidase reduced ceramide levels and conferred resistance to DTIC. In conclusion, we report that increased levels of ceramide, due to enhanced degradation of aCDase, are in part responsible for the cell death effects of DTIC. These results suggest that down-regulation of aCDase alone or in combination with DTIC may represent a useful tool in the treatment of metastatic melanoma.     

518A2 melanoma cells are protected by G3139 and other antineoplastic agents against the cytotoxic effects of DTIC

G3139 is an antisense Bcl-2 phosphorothioate oligonucleotide that has been combined with DTIC in a phase III clinical trial in melanoma. However, its actual mechanism of action in melanoma is controversial. Treatment of 518A2 melanoma cells with either G3139 or G4126 (a two-base mismatch) and then with light-activated DTIC caused these cells (but not SK-Mel-30 or 346.1 cells) to be protected against the cytotoxic effects of DTIC. This cytoprotection was not recapitulated with a phosphodiester congener of G3139 nor with a small interfering RNA (siRNA) also targeted to the Bcl-2 mRNA. Administering the drugs in reverse order also did not produce cytoprotection, and an 18- mer phosphorothioate homopolymer of thymidine was also inactive. Subsequently, it was discovered that gemcitibine and cis-platinum also induced cytoprotection to DTIC in this cell line, suggesting that the cytoprotection is a stress response to chemical proapoptotic stress. Cytoprotection was completely inhibited by O(6)-benzylguanine, an inhibitor of O(6)-guanosine alkyltransferase (OGAT) activity. However, a direct assay of OGAT activity demonstrated that 518A2 melanoma cells are essentially completely devoid of it, either basally or induced. The cytoprotection may thus be caused by a chemical stress-induced increase in mismatch repair activity.     

Increased immunogenicity of L1210 leukemia following short-term exposure to 5(3,3′-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) in vivo or in vitro

Summary Short-term exposure of L1210 Ha leukemia to DTIC in vivo or in vitro resulted in the generation of leukemic cells that were moderately immunogenic for histocompatible (BALB/C × DBA/2)F₁ (CD2F₁) mice. In vivo treatment was carried out in the peritoneal cavity of CD2F₁ host for 8–36 h. In vitro experiments were performed in glass vessels, in which tumor cells were incubated with DTIC for 2 h at 37° C. The in vitro generation of immunogenic leukemia was conditioned by the presence of mouse liver microsomes capable of producing metabolic transformation of DTIC. It follows that the increase of tumor cell immunogenicity produced in vitro and possibly in vivo by DTIC is due to (a) metabolic product (s) that has (have) not yet been identified. Somatic mutation, selection, viral activation, or other mechanisms could be responsible for the DTIC effect. The present studies suggest that similar in vivo or in vitro techniques could be used to obtain human tumor cells with higher immunogenicity.Abbreviations AIC 5-aminoimidazole-4-carboxamide - BCNU 1,3-bis-chloroethyl nitrosourea - Cy of cyclophosphamide - DMITI drug-mediated increase tumor immunogenicity - DTIC 5(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide - MLP mouse liver preparation - PB phenobarbital     

Acute toxic effects of single dose dacarbazine: hematological and histological changes in an animal model

Treatment of advanced soft tissue sarcoma usually includes dacarbazine (DTIC), an alkylating agent that methylates DNA and is active during all phases of the cell cycle. Common side effects of DTIC include nausea, vomiting, impaired liver and kidney function, myelosuppression, and pneumonia. There are no accounts, however, of histological and hematological changes caused by DTIC. We investigated acute hematological and morphological changes in different organs and in tumors that were caused by a single dose of DTIC. Adult Syrian golden hamsters were inoculated with a suspension of tumorigenic baby hamster kidney (BHK) cells by subcutaneous injection. On day 14 after inoculation, doses of 1.4, 1.6, 1.8 or 2.0 g/m² DTIC were injected intraperitoneally into the hamsters. Hamsters in the control group were injected with physiological saline in the same way. Seven days after drug or saline injection the animals were sacrificed and samples of blood, heart, kidney, liver, lungs, spleen, small intestine and tumor were excised, processed and analyzed. Mitoses were counted using an ocular extension with engraved frame. Anemia, thrombocytopenia and leukocytosis were found in the control group of hamsters with fibrosarcoma, whereas animals with fibrosarcoma treated with DTIC developed anemia, thrombocytopenia and leukopenia. Severe pneumonia and moderate hepatitis were detected in all DTIC treated groups. Effects of DTIC on tumor cells included rounding and enlargement of nuclei and rarefaction of chromatin. The number of mitoses was reduced with increasing doses of DTIC. Hepatitis, myelosuppression, pneumonia, and dose-related inhibition of tumor cell proliferation were observed after a single dose of DTIC.     

Adoptive immunity in mice challenged with L1210/DTIC clones

Summary New antigenic specificities, not detectable on parental cells, have been induced by many investigators in mouse lymphomas by treatment with the antitumor agent 5(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC). The antigens are transmissible, after withdrawal of the drug treatment, as an inheritable character. The mechanism of induction, the molecular nature, and the number of the new antigenic specificities have not been completely elucidated. Four clones from murine leukemia L1210 isolated and expanded in vitro were treated in vivo with DTIC and the new sublines were studied in detail. The four drug-treated sublines studied exhibited strong immunogenicity since they were rejected by syngeneic animals. Immunosuppressed animals challenged with 10⁷ A/DTIC or P/DTIC cells were reciprocally protected by the adoptive transfer of spleen cells from donors that had rejected a lethal challenge of A/DTIC or P/DTIC clones. In a similar fashion, the adoptive transfer of spleen cells obtained from animals that had rejected the Q/DTIC or the R/DTIC clones protected immunosuppressed mice challenged with Q/DTIC or R/DTIC cells. No antitumor activity was observed in cross-protective schedules other than those indicated. It was been concluded that (a) the L1210 leukemia line does not have antigenic cells, (b) four DTIC-treated clone sublines were rejected by compatible hosts, and (c) two mutually exclusive sets of antigens were expressed in four antigenic clone sublines.Research supported in part by P.F.O. Contract Grant from C. N. R., Rome, Italy     

Mechanisms of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (Dacarbazine) cytotoxicity toward Chinese hamster ovary cells in vitro are dictated by incubation conditions

Decomposition of the antitumor agent 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC, Dacarbazine) produces several potentially toxic compounds, the concentration of which depend on incubation parameters such as pH, temperature and illumination. The action of DTIC on chinese hamster ovary (CHO) cell clone formation in the dark (7-8-day incubation) reflects the slow formation of 2-azahypoxanthine. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8)-deficient cells are resistant to DTIC under these conditions, reflecting their inability to utilize 2-azahypoxanthine. The toxicity of DTIC in conventional survival experiments (1-2-h exposure to drug) is dependent upon illumination and is highly influenced by the pH of the medium. Toxicity of DTIC in these experiments appears to reflect rapid accumulation of the immediate photodecomposition product of the drug, 4-diazoimidazole-5-carboxamide (DZC), since HGPRT-deficient cells are not resistant to DTIC under these conditions. The biologically initiated pathway of DTIC action (enzymatic hydroxylation) has little, if any, role in the action of this agent toward cultured CHO cells.     

Antigenic changes of a murine lymphoma by in vivo treatment with triazene derivatives

Summary Five aryltriazenes were studied for efficacy in mediating immunogenic changes of tumor cells by in vivo treatment of lymphoma-bearing mice. It was found that four analog compounds produced increase in cell immunogenicity similar to that described for 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC), one of the five being by contrast completely inactive. Moreover, the use of a drug-resistant lymphoma illustrates that cytotoxic activity is not mandatory for the appearance of the immunogenic changes.The results show that a drug-mediated increase of tumor immunogenicity (DMITI) can be induced by triazene derivatives not containing the imidazole moiety.     

Antitumor and antimetastatic effects of dacarbazine combined with cyclophosphamide and interleukin-2 in Lewis lung carcinoma (3LL)

The antitumor and antimetastatic activity of dacarbazine (DTIC) alone or in combination with cyclophosphamide (CY) was tested in C57BL/6 mice bearing Lewis lung carcinoma (3LL). Treatment with both agents significantly reduced tumor growth and the number of metastases. These effects were associated with marked changes of the biochemical and immunological properties of drug-treated 3LL cells, i.e. (a) reduction of α6 integrin expression, (b) increased susceptibility to natural immunity in vivo, as measured in terms of rapid clearance from mouse lungs of prelabeled 3LL cells injected i.v. and (c) increased immunogenicity, as assessed by T-cell-mediated immune responses (i.e. graft rejection by intact syngeneic mice, and frequency of specific CTL precursors recognizing DTIC/CY-treated cancer cells). The immunotherapeutic advantage afforded by increased immunosensitivity and immunogenicity of 3LL cells exposed to DTIC+CY appears to be markedly reduced in vivo by the profound immunodepressive effects of these drugs. Within this context, addition of interleukin-2 was found to increase the antitumor and antimetastatic activity of this chemotherapeutic regimen. The present study shows, for the first time in a solid tumor model, that a biological response modifier increases the antitumor efficacy of drugs that are able to affect the immunological properties of cancer cells.     

Antitumor and antimetastatic effects of dacarbazine combined with cyclophosphamide and interleukin-2 in Lewis lung carcinoma (3LL)

The antitumor and antimetastatic activity of dacarbazine (DTIC) alone or in combination with cyclophosphamide (CY) was tested in C57BL/6 mice bearing Lewis lung carcinoma (3LL). Treatment with both agents significantly reduced tumor growth and the number of metastases. These effects were associated with marked changes of the biochemical and immunological properties of drug-treated 3LL cells, i.e. (a) reduction of 6 integrin expression, (b) increased susceptibility to natural immunity in vivo, as measured in terms of rapid clearance from mouse lungs of prelabeled 3LL cells injected i.v. and (c) increased immunogenicity, as assessed by T-cell-mediated immune responses (i.e. graft rejection by intact syngeneic mice, and frequency of specific CTL precursors recognizing DTIC/CY-treated cancer cells). The immunotherapeutic advantage afforded by increased immunosensitivity and immunogenicity of 3LL cells exposed to DTIC+CY appears to be markedly reduced in vivo by the profound immunodepressive effects of these drugs. Within this context, addition of interleukin-2 was found to increase the antitumor and antimetastatic activity of this chemotherapeutic regimen. The present study shows, for the first time in a solid tumor model, that a biological response modifier increases the antitumor efficacy of drugs that are able to affect the immunological properties of cancer cells.     

Chemical xenogenization of murine lymphoma cells with triazene derivatives: Immunotoxicological studies

Summary Equitoxic doses of 5-(3-3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) and aryl-triazene derivatives (compounds all capable of inducing a marked increase in murine tumor cell immunogenicity) were studied for their effects on the host immune system. At different times after drug exposure the animals were tested for allograft responses, competence in producing lymphocytes active in lethal graft-versus-host disease, delayed-type hypersensitivity, humoral antibody production, and mitogen responsiveness. While some of the aryl-triazenes tested (DM-COOK DM-NO₂) showed a pattern of immunodepression similar to that of DTIC, others were less (MIC, MM-COOK, MM-Cl) or far less (DM-Cl, MM-NO₂) active than DTIC in impairing host immunocompetence, although all retained or even augmented their ability to induce chemical xenogenization.This work was supported by Progetto Finalizzato Controllo della Crescita Neoplastica contracts no. 83.00815.96 and no. 83.00838.96 (CNR, Rome, Italy)     

Chemoimmunotherapy for melanoma with dacarbazine and 2,4-dinitrochlorobenzene elicits a specific T cell-dependent immune response

An empirically established chemoimmunotherapy for metastatic melanoma combines the systemic administration of the chemotherapeutic agent dacarbazine (DTIC) with the epifocal application of the contact sensitizer 2,4-dinitrochlorobenzene (DNCB) on cutaneous metastases. Although this therapy yields high response rates resulting in prolonged survival, the mechanisms involved remain unknown. Here, we investigated whether treatment of tumor-bearing mice with DTIC and DNCB resulted in a specific immune response against the tumor. Subcutaneous (s.c.) tumors and lung metastases were induced in C57BL/6 mice by injecting syngeneic B16-melanoma cells s.c. or into the lateral tail vein, respectively. Mice were treated with intraperitoneal injections of DTIC followed by epifocal application of DNCB. This therapeutic approach significantly reduced the growth of s.c. tumors as well as lung metastases. Our data showed that the effector mechanisms involved are dependent on T cells. No therapeutic effect was observed in immunodeficient RAG-1(-/-) mice, or when the contact sensitizer DNCB was replaced by skin irritants (croton oil or tributyltin). Splenic lymphocytes obtained from treated mice displayed a three-fold higher specific cytolytic activity against B16 cells than in tumor-bearing controls. Both CD8(+) and CD4(+) T cells were able to lyse B16 cells. No changes were observed in natural killer (NK) cell activity. Likewise, tumor-infiltrating lymphocytes (TIL) of treated mice showed higher cytolytic activity than that of controls. Analysis of cytokine expression in s.c. tumors revealed increased mRNA levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) in treated tumors. Together, these findings demonstrate the ability of DTIC/DNCB treatment to induce an effective T cell-dependent host immune response against a syngeneic tumor.     

Treatment of melanoma with 5-fluorouracil or dacarbazine in vitro sensitizes cells to antigen-specific CTL lysis through perforin/granzyme- and Fas-mediated pathways

Several factors may influence sensitivity of melanoma cells to CTL lysis. One is the avidity of the CTL TCR. A second is that certain cytotoxic drugs have been reported to sensitize cancer cells to CTL lysis through Fas-mediated apoptosis. In this study, we examined whether antineoplastic agents 5-fluorouracil (5-FU) and dacarbazine (DTIC) sensitize melanoma cells to lysis of G209 peptide-specific CTL. Our results show that CTL generated from PBMC are HLA-A2 restricted and gp100 specific. Treatment with 5-FU or DTIC sensitized melanoma cells to lysis of G209-specific CTL. Most importantly, 5-FU- or DTIC-treated melanoma cells also became sensitive to low-avidity CTL, which per se are less cytolytic to melanomas. We sought to identify apoptotic pathways mediating this effect. The enhanced cytolysis was mediated through the perforin/granzyme pathway. Although 5-FU up-regulated FasR expression on melanoma cells, sensitization was not blocked by anti-Fas Ab, and the G209-specific CTL was Fas ligand (FasL) negative. However, when G209-specific CTL were stimulated to express FasL, FasL signaling also contributed to enhanced cytolysis. DTIC treatment, which did not increase FasR expression, also sensitized FasL-mediated killing induced by neutralizing anti-Fas Ab. For CD95L-positive G209-specific CTL, the sensitization was primarily mediated through the perforin/granzyme pathway regardless of up-regulation of FasR. The findings demonstrate that cytotoxic drug-mediated sensitization primes both perforin/granzyme and Fas-mediated killing by melanoma-specific CTL. Considering that most of autoreactive antitumor CTL are low avidity, the findings provide experimental basis for understanding cytotoxic and immunologic therapeutic synergy in melanoma.     

Cytotoxicity studies of human melanoma cells and fibroblasts.

In seven human melanoma cell lines and one human fibroblast strain some correlation of resistance to cell killing was found with two bifunctional alkylating agents (melphalan, chlorambucil) and three monofunctional agents (4(5)-(3,3-dimethyl-l-triazeno)imidazole-5(4)-carboxamide (DTIC), methylmethane sulphonate (MMS) and N-methyl-N1-nitro-N-nitrosoguanidine (MNNG), but little cross-resistance was found between these two groups of agents or with cytosine arabinoside (ara-C). In contrast to previous studies with rodent tumours, potentially synergistic (chloroquine, arginine) or antagonistic (ascorbic acid, leucine) compounds did not affect the toxicity of melphalan in a human melanoma cell line. In two melanoma lines DTIC induced patterns of DNA damage (inhibition of semi-conservative synthesis) and repair (strand breaks and repair synthesis) similar to, but not identical with, those induced by the methylating agent MMNG. These results suggest that a methylating species is derived from DTIC but has a different reactivity toward DNA compared with MNNG.     

Dacarbazine and the Agonistic TRAIL Receptor-2 Antibody Lexatumumab Induce Synergistic Anticancer Effects in Melanoma

Mapatumumab and lexatumumab (targeting death receptor 4 (DR4) and 5 (DR5), respectively) are agonistic TRAIL receptor antibodies that induce apoptosis in a wide range of cancer cells. The potency of mapatumumab and lexatumumab was assessed in mono therapy protocols, and the ability to sensitize for dacarbazine (DTIC) treatment was explored in ten different melanoma cell lines. Our data indicated that melanoma cell lines tend to be resistant to mapatumumab, most likely due to low expression of DR4, while a dose dependent response to lexatumumab was observed. Combining DTIC and lexatumumab induced an additive or synergistic effect on cell death in the various melanoma cell lines. The synergistic effect observed in the FEMX-1 cell line was related to enhanced cleavage of Bid in parallel with elevated expression of the pro-apoptotic proteins Bim, Bax and Bak. Furthermore, the anti-apoptotic proteins Bcl-XL, cIAP-1, XIAP and livin were down regulated. Cleavage of Bid and down regulation of cIAP-2 and livin were observed in vivo. Altogether, these data suggest a change in the balance between pro- and anti-apoptotic proteins favoring induction of apoptosis. In the more therapy resistant cell line, HHMS, no changes in the pro- and anti-apoptotic proteins were observed. FEMX-1 xenografts treated with DTIC and lexatumumab showed reduced growth and increased level of apoptosis compared to the control groups, providing arguments for further evaluation of this combination in melanoma patients.     

Metformin pretreatment reduces effect to dacarbazine and suppresses melanoma cell resistance

Oxidative stress role on metformin process of dacarbazine (DTIC) inducing resistance of B16F10 melanoma murine cells are investigated. To induce resistance to DTIC, murine melanoma cells were exposed to increasing concentrations of dacarabazine (DTIC-res group). Metformin was administered before and during the induction of resistance to DTIC (MET-DTIC). The oxidative stress parameters of the DTIC-res group showed increased levels of malondialdehyde (MDA), thiol, and reduced nuclear p53, 8-hydroxy-2?-deoxyguanosine (8-OH-DG), nuclear factor kappa B (NF-?B), and Nrf2. In presence of metformin in the resistant induction process to DTIC, (MET-DTIC) cells had increased antioxidant thiols, MDA, nuclear p53, 8-OH-DG, Nrf2, and reducing NF-?B, weakening the DTIC-resistant phenotype. The exclusive administration of metformin (MET group) also induced the cellular resistance to DTIC. The MET group presented high levels of total thiols, MDA, and reduced percentage of nuclear p53. It also presented reduced nuclear 8-OH-DG, NF-?B, and Nrf2 when compared with the control. Oxidative stress and the studied biomarkers seem to be part of the alterations evidenced in DTIC-resistant B16F10 cells. In addition, metformin administration is able to play a dual role according to the experimental protocol, preventing or inducing a DTIC-resistant phenotype. These findings should help future research with the aim of investigating DTIC resistance in melanoma.     

Experimental models of immunochemotherapeutic synergism: study of the influence of the treatment schedule

Graded doses of LSTPA or L1210 leukemia cells were injected ip or iv into fully compatible hosts or mice incompatible for Multiple Minor Histocompatibility Loci (MMHL). Three days later the animals were treated with single doses of BCNU, NM, DTIC and VCR. The results showed that NM and VCR could synergize with the weak anti-tumor immune responses of MMHL-histocompatible mice only upon ip injection of the tumor. If the same tumor has been injected iv, only BCNU could synergize with the host's antitumor response. On DTIC treatment, no synergistic effects were detectable for either route of tumor's challenge.     

Curcumin Analog DM-1 in Monotherapy or Combinatory Treatment with Dacarbazine as a Strategy to Inhibit In Vivo Melanoma Progression

Malignant melanoma is a highly aggressive form of skin cancer with a high mortality rate if not discovered in early stages. Although a limited number of treatment options for melanoma currently exist, patients with a more aggressive form of this cancer frequently decline treatment. DM-1 is a sodium phenolate and curcumin analog with proven anticancer, anti-proliferative and anti-metastatic properties. In this paper, the DM-1 compound showed in vivo antitumor activity alone or in combination with chemotherapeutic DTIC in B16F10 melanoma-bearing mice. Beneficial effects such as melanoma tumor burden reduction with pyknotic nuclei, decreased nuclei/cytoplasmic ratio and nuclear degradation occurred after DM-1 treatment. No toxicological changes were observed in the liver, kidneys, spleen and lungs after DM-1 monotherapy or DTIC combined therapy. DTIC+DM-1 treatment induced the recovery of anemia arising from melanoma and immunomodulation. Both DM-1 treatment alone and in combination with DTIC induced apoptosis with the cleavage of caspase-3, -8 and -9. Furthermore, melanoma tumors treated with DM-1 showed a preferential apoptotic intrinsic pathway by decreasing Bcl-2/Bax ratio. Considering the chemoresistance exhibited by melanoma towards conventional chemotherapy drugs, DM-1 compound in monotherapy or in combination therapy provides a promising improvement in melanoma treatment with a reduction of side effects.     

Immunoregulatory protein B7‐H3 promotes growth and decreases sensitivity to therapy in metastatic melanoma cells

B7‐H3 (CD276) belongs to the B7 family of immunoregulatory proteins and has been implicated in cancer progression and metastasis. In this study, we found that metastatic melanoma cells with knockdown expression of B7‐H3 showed modest decrease in proliferation and glycolytic capacity and were more sensitive to dacarbazine (DTIC) chemotherapy and small‐molecule inhibitors targeting MAP kinase (MAPK) and AKT/mTOR pathways: vemurafenib (PLX4032; BRAF inhibitor), binimetinib (MEK‐162; MEK inhibitor), everolimus (RAD001; mTOR inhibitor), and triciribidine (API‐2; AKT inhibitor). Similar effects were observed in melanoma cells in the presence of an inhibitory B7‐H3 monoclonal antibody, while the opposite was seen in B7‐H3‐overexpressing cells. Further, combining B7‐H3 inhibition with small‐molecule inhibitors resulted in significantly increased antiproliferative effect in melanoma cells, as well as in BRAF^V600E mutated cell lines derived from patient biopsies. Our findings indicate that targeting B7‐H3 may be a novel alternative to improve current therapy of metastatic melanoma.