Evaluation of cellular immunological responses in mono- and polymorphic clinical forms of post-kala-azar dermal leishmaniasis in India.

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic dermal complication that occurs usually after recovery from visceral leishmaniasis (VL). The disease manifests into macular, papular and/or nodular clinical types with mono- or polymorphic presentations. Here, we investigated differences in immunological response between these two distinct clinical forms in Indian PKDL patients. Peripheral blood mononuclear cells of PKDL and naive individuals were exposed in vitro to total soluble Leishmania antigen (TSLA). The proliferation index was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based lymphoproliferative assay. Cytokines and granzyme B levels were determined by cytometric bead array. Parasite load in tissue biopsy samples of PKDL was quantified by quantitative polymerase chain reaction (qPCR). The proportion of different lymphoid subsets in peripheral blood and the activated T cell population were estimated using flow cytometry. The study demonstrated heightened cellular immune responses in the polymorphic PKDL group compared to the naive group. The polymorphic group showed significantly higher lymphoproliferation, increased cytokines and granzyme B levels upon TSLA stimulation, and a raised proportion of circulating natural killer (NK) T cells against naive controls. Furthermore, the polymorphic group showed a significantly elevated proportion of activated CD4(+) and CD8(+) T cells upon in-vitro TSLA stimulation. Thus, the polymorphic variants showed pronounced cellular immunity while the monomorphic form demonstrated a comparatively lower cellular response. Additionally, the elevated level of both activated CD4(+) and CD8(+) T cells, coupled with high granzyme B secretion upon in-vitro TSLA stimulation, indicated the role of cytotoxic cells in resistance to L. donovani infection in polymorphic PKDL.

Potentiation of lonidamine and diazepam, two agents acting on mitochondria, in human glioblastoma treatment.

BACKGROUND: Cellular metabolism in glioblastoma multiforme, the most common primary brain tumor in humans, is characterized by a high rate of aerobic glycolysis that is dependent on mitochondria-bound hexokinase. Moreover, high levels of glucose utilization and tumor aggressiveness in glioblastoma are associated with a high density of mitochondrial benzodiazepine receptors. We sought to inhibit glioblastoma metabolism by simultaneously inhibiting hexokinase with lonidamine and binding benzodiazepine receptors with diazepam. METHODS: Cellular glioblastoma metabolism in five glioblastoma cell lines was assessed in vitro by measuring cell proliferation (by use of a tetrazolium-based colorimetric assay, measurement of DNA synthesis, and assessment of cell cycle distribution), by measuring membrane fluidity (by fluorescence polarization measurement of cells stained with a fluorescent probe), and by measuring changes in intracellular pH. Immunodeficient nude mice bearing subcutaneous xenografts of human glioblastoma cells were used to assess the antitumor activities of lonidamine and diazepam; the mice were treated twice daily with lonidamine (total daily dose of 160 mg/kg body weight) and/or diazepam (total daily dose of 1 mg/kg body weight) for 10 consecutive days. RESULTS: When used in combination, the two drugs had a stronger effect on glioblastoma cell proliferation and metabolism in vitro than did either agent used alone. In vivo, the combination of lonidamine and diazepam was significantly more effective in reducing glioblastoma tumor growth than either drug alone (two-sided P<.01, Mann-Whitney U test, comparing growth of treated tumors with that of untreated tumors); this tumor growth retardation was maintained as long as treatment was given. CONCLUSION: The combination of lonidamine and diazepam--drugs that target two distinct mitochondrial sites involved in cellular energy metabolism--potentiates the effects of the individual drugs and may prove useful in the treatment of human glioblastomas.

Methionine deprivation and methionine analogs inhibit cell proliferation and growth of human xenografted gliomas.

Growth of numerous malignant tumors depends on an exogenous methionine (MET) supply, while endogenously synthesized MET supports normal cell proliferation. Because an antitumor effect should be obtained by aggravating the altered MET metabolism in gliomas, MET dependency of human xenografted gliomas was evaluated and a therapeutic approach using MET deprivation or MET analogs to induce MET starvation was applied. In vitro proliferation inhibition of glioma cell lines by MET deprivation and two MET analogs, ethionine (ETH) and trifluoromethylhomocysteine (TFH), was measured. Proliferation of 7 human glioma cell lines tested was inhibited in MET-free medium, and was poorly or not reversed by homocysteine (HCY). ETH or TFH (concentration range: 0.005-2 mg/ml) inhibited proliferation of all cell lines tested. MET analog-induced inhibition was abolished by MET and enhanced by HCY. Cell-cycle alterations due to MET deprivation were optimally assessed after 30 h of culture and bromodeoxyuridine incorporation. In MET- medium, cells were arrested in the G1-phase. ETH induced a dramatic accumulation of cells in the G2-phase. ATP contents were reduced by MET analogs only in HCY+ medium, suggesting complementary effects of MET analogs and HCY. Human glioma bearing nude mice were fed an amino acid-substituted MET- HCY-supplemented diet (MET-HCY+) and/or treated with MET analogs, injected intraperitoneally daily. Using two human xenografted tumors derived from gliomas, antitumor effects were obtained by subjecting tumor-bearing nude mice to MET starvation. TG-1-MA was more sensitive to MET depletion (40% of growth inhibition, P < 0.10) than TG-8-OZ (no growth inhibition). Antitumor effects of a MET-HCY+ diet and 200 mg/kg of ETH were potentiated when co-administered to glioma-bearing mice (77% GI, P < 0.025 and 67%, P < 0.0057 to TG-1-MA and TG-8-OZ respectively). A dose-response effect with no toxicity was obtained when the ETH dose was increased 10 fold. Potentiation of the effects of ETH and a MET-free diet indicates that they probably act on the same pathway but not the same target. In conclusion, experimentally induced MET deprivation and MET-analog treatment retarded the growth of human gliomas. Combination of MET-analog therapy with MET substitution by HCY enhanced their respective effects.

Response to 5-fluorouracil of orthotopically xenografted human colon cancers with a microsatellite instability: influence of P53 status.

Response to 5-FU of 5 different human colon cancers (hCC) with a DNA microsatellite instability (MSI), xenografted into nude mice, was analysed according to their p53 status. Two hCC (TC82 and TC71) had a mutated p53 (mutp53), while two others (TC33 and LoVo) had a wild type p53 (wtp53); the fifth tumour (X17LoVo) originated from the stable transfection of LoVo cells with a dominant mutp53 (273his) vector. All tumours were implanted onto the caecum of nude mice to induce orthotopic growth of hCC. 5-FU was administered at 40 mg/kg per day for 5 consecutive days. A significant growth inhibition of TC82 and TC71 tumours (of 68 and 60%, p < 0.05 and < 0.01 respectively) was observed, whereas 5-FU had no effect on TC33 and LoVo. Moreover, the mutp53 transfected tumour, X17LoVo, displayed significant sensitivity to 5-FU (p < 0.05). This suggests that distinct genetic alterations influence differently the response of hCC to this antimetabolite.

Topoisomerase I-DNA covalent complexes in human colorectal cancer xenografts with different p53 and microsatellite instability status: relation with their sensitivity to CTP-11.

The topoisomerase I poison CPT-11 has proved efficient for the treatment of untreated metastatic colorectal cancers (CRC) and those refractory to fluoropyrimidines. However, the interpatient variability is important. A previous in vitro study suggested that measurements of the level of topoisomerase I-DNA intermediates trapped by camptothecin may be useful to estimate the chemosensitivity of colon carcinoma cell lines. To verify this hypothesis, we developed an immuno-assay to detect covalent topoisomerase I-DNA complexes in a series of human colorectal cancers xenografted in nude mice. Six human CRCs were selected for their distinctive p53 and microsatellite instability (MSI) status. Tumour lysates, prepared from mice untreated or treated with CPT-11, were fractionated onto CsCl gradients to separate free and DNA-bound topoisomerase I by centrifugation. Interestingly, significant levels of DNA-topoisomerase I complexes were detected in the tumours most responsive to the treatment with CPT-11, irrespective of their MSI and p53 phenotypes. Our in vivo study fully agrees with the predictions from the in vitro data indicating that evaluation of topoisomerase I-DNA complexes would be useful to predict the response of CRC to a treatment with CPT-11.

Specific cpb copies within the Leishmania donovani complex: evolutionary interpretations and potential clinical implications in humans.

Leishmania infantum and Leishmania donovani both pertain to the L. (L.) donovani complex and are responsible for visceral leishmaniasis. To explore the L. donovani complex, we focused our study on cysteine protease B (cpb) and especially on 2 cpb copies: cpbE and cpbF. We selected cpb genes because of their phylogenetic interest and host-parasite interaction involvement. Sequencing these 2 copies revealed (i) that cpbE is specific to L. infantum and cpbF is specific to L. donovani and (ii) that these 2 copies are different in length and sequence. Phylogenetic analysis and protein predictions were carried out in order to compare these copies (i) with other trypanosomatid cpb, especially L. mexicana, and (ii) within the L. donovani complex. Our results revealed patterns specific to the L. donovani complex such as the COOH-terminal extension, potential epitopes and N-glycosylation sites. Moreover, phylogenetic analysis revealed different levels of polymorphism between L. infantum and L. donovani and confirmed the ancestral status of the latter. L. infantum has a shorter sequence and a deleted sequence responsible for modifications in protein conformation and catalytic triad. Considering the clinical aspect, L. infantum dermotropic strains appeared more polymorphic than L. infantum viscerotropic strains.

Phenotypical characteristics, biochemical pathways, molecular targets and putative role of nitric oxide-mediated programmed cell death in Leishmania.

Nitric oxide (NO) has been demonstrated to be the principal effector molecule mediating intracellular killing of Leishmania, both in vitro and in vivo. We investigated the type of cell death process induced by NO for the intracellular amastigote stage of the protozoa Leishmania. Specific detection methods revealed a rapid and extensive cell death with morphological features of apoptosis in axenic amastigotes exposed to NO donors, in intracellular amastigotes inside in vitro - activated mouse macrophages and also in activated macrophages of regressive lesions in a leishmaniasis-resistant mouse model. We extended our investigations to the dog, a natural host-reservoir of Leishmania parasites, by demonstrating that co-incubation of infected macrophages with autologous lymphocytes derived from dogs immunised with purified excreted-secreted antigens of Leishmania resulted in a significant NO-mediated apoptotic cell death of intracellular amastigotes. From the biochemical point of view, NO-mediated Leishmania amastigotes apoptosis did not seem to be controlled by caspase activity as indicated by the lack of effect of cell permeable inhibitors of caspases and cysteine proteases, in contrast to specific proteasome inhibitors, such as lactacystin or calpain inhibitor I. Moreover, addition of the products of two NO molecular targets, cis-aconitase and glyceraldehyde-3-phosphate dehydrogenase, also had an inhibitory effect on the cell death induced by NO. Interestingly, activities of these two enzymes plus 6-phosphogluconate dehydrogenase, parasitic enzymes involved in both glycolysis and respiration processes, are overexpressed in amastigotes selected for their NO resistance. This review focuses on cell death of the intracellular stage of the pathogen Leishmania induced by nitrogen oxides and gives particular attention to the biochemical pathways and the molecular targets potentially involved. Questions about the role of Leishmania amastigotes NO-mediated apoptosis in the overall infection process are raised and discussed.

Growth of methionine-dependent human prostate cancer (PC-3) is inhibited by ethionine combined with methionine starvation.

Methionine (MET) is required for cell metabolism. MET endogenously synthesized from homocysteine (HCY) supports the proliferation of normal cells, but not that of numerous malignant cells, as shown previously. MET starvation should have an anti-tumour effect, and its deleterious effects on the hosts might be prevented by HCY. Anti-tumour effects of MET starvation must be reinforced by ethionine (ETH), a MET analogue. MET dependency of PC-3, a human prostate cancer cell line, was studied in vitro. Proliferation of PC-3 cells, cultivated in MET-free medium, was 29% compared with growth in MET+HCY- medium. Addition of HCY to MET-free medium increased the proliferation rate to 56%. The concentration of ETH required to decrease the PC-3 cell proliferation rate to 50% (IC50) was 0.5 mg ml(-1) in MET-HCY- medium. ETH-induced inhibition was abolished by MET addition and was reinforced by HCY. PC-3 cell cycle was blocked in the S-G2-phase after 30 h culture in the absence of MET; this blockage was not reversed by addition of HCY. ETH at the IC50 in MET-HCY+ medium blocked DNA replication. Apoptotic cells appeared after 30 h incubation in MET-HCY+ medium only when ETH was added. ATP pools were decreased after 15 h of culture in MET-free medium. In vivo, MET starvation was obtained by feeding tumour-bearing mice a diet containing a synthetic amino acid mixture as the protein supply, in which HCY replaced MET. Given to nude mice bearing xenografted PC-3, from day 1 after grafting and for 3 weeks, this diet inhibited tumour growth (34% on day 20, P < 0.007); this effect was potentiated by ETH (200 mg kg(-1) day(-1) i.p.) (56% on day 20, P < 5 x 10(-5)). The differences between the effects of these two treatments were significant (P < 0.017) and optimal on day 20. These data showed that combination of ETH and HCY slowed the proliferation of prostate cancer cells in vitro and in vivo, decreased ATP synthesis and caused cell cycle arrest and apoptosis. Experimental therapy based on cancer cell MET metabolism deficiency could be efficient for treating advanced prostate cancers refractory to current therapies.

Single alteration of p53 or E-cadherin genes can alter the surgical resection benefit in an experimental model of colon cancer.

PURPOSE: p53 and E-cadherin mutations are associated with a high risk of metastatic potential and local recurrence after colorectal surgery. LoVo, a human colon cancer cell line expressing a wild-type p53 and a normal E-cadherin, was studied. Clone LoVo-XC17 was obtained from LoVo cells transfected with a vector bearing a p53 273his mutation. Clone LoVo-92R4 was obtained from LoVo by culture cells with an E-cadherin down-regulation. LoVo, LoVo-XC17, and LoVo-92R4 were studied for in vivo behavior in a surgical intracolonic graft model. METHODS: Ten nude mice were used per cell line. A colonic tumor was obtained by tumor implantation into the cecal wall. The cecal tumor was resected at Day 15; at this time the volumes of the different tumors were similar. RESULTS: Surgical resection of the LoVo tumor led to 100 percent disease-free animals at one month. Surgical resection of mice grafted with the LoVo-XC17 line did not cure any mice (0/10; P = 0.001). Mice had local recurrences (10/10), mesenteric lymph node metastases (9/10), liver metastases (2/10), and peritoneal carcinomatosis (8/10). Surgical resection of LoVo-92R4 tumors led to cures in 30 percent (3/10), whereas 70 percent had isolated mesenteric lymph node metastases (7/10; P = 0.003). CONCLUSION: In this model surgical tumor resection was consistently effective for colonic tumors with functional p53 and E-cadherin, it was consistently ineffective with tumors displaying a mutated p53, and it was partially effective with E-cadherin-deficient tumors. This study shows that the alteration of a single gene can be associated with a profound alteration of surgical resection benefit.

Sensitivity to CPT-11 of xenografted human colorectal cancers as a function of microsatellite instability and p53 status.

Biological parameters influencing the response of human colorectal cancers (CRCs) to CPT-11, a topoisomerase 1 (top1) inhibitor, were investigated using a panel of nine CRCs xenografted into nude mice. CRC xenografts differed in their p53 status (wt or muf) and in their microsatellite instability phenotype (MSI+ when altered). Five CRC xenografts were established from clinical samples. All five had a functional p53, two were MSI+ and three were MSI-. Tumour-bearing nude mice were treated intraperitonealy (i.p.) with CPT-11. At 10 mg kg(-1) of CPT-11, four injections at 4-day intervals, four of the five xenografts responded to CPT-11 (growth delay of up to 10 days); the non-responder tumour was MSI-. At 40 mg kg(-1) of CPT-11, six injections at 4-day intervals, the five CRCs displayed variable but marked responses with complete regressions. In order to assess the role of p53 status in CPT-11 response, four CRC lines were used. HT29 cell line was MSI-/Ala273-mutp53, its subclone HT29A3 being transfected by wtp53. LoVo cell line was MSI+/wtp53, its subclone X17LoVo dominantly expressed Ala273-mutp53 after transfection. LoVo tumours (MSI+/mutp53) were more sensitive than X17LoVo (MSI+/mutp53. HT 29 tumours (MSI-Imutp53), were refractory to CPT-11 while HT29A3 tumours (MSI-/wtp53) were sensitive, showing that wtp53 improves the drug-response in these MSI- tumours. Levels of mRNA expression of top1, fasR, TP53 and mdr1 were semi-quantified by reverse transcription polymerase chain reaction. None of these parameters correlated with CPT-11 response. Taken together, these observations indicate that MSI and p53 alterations could be associated with different CPT-11 sensitivities; MSI phenotype moderately influences the CPT-11 sensitivity, MSI+ being more sensitive than MSI(-)CRC freshly obtained from patients, mutp53 status being associated with a poor response to CPT-11.

Synergistic efficacy of 3n-butyrate and 5-fluorouracil in human colorectal cancer xenografts via modulation of DNA synthesis.

BACKGROUND & AIMS: Butyrate, produced in the colon lumen, maintains mucosal cell homeostasis. Poorly diffusible, its access is compromised in growing colon cancers and absent in distant metastases. Butyrate regulates DNA synthesis. We postulated that systemic administration of butyrate should reduce colon cancer growth and enhance 5-fluorouracil (5-FU) efficacy. METHODS: A stable derivative of butyrate (3n-But) was used. The antitumoral efficacy of 5-FU and 3n-But, alone or combined, was evaluated in human colorectal cancers (hCRCs) subcutaneously, orthotopically, or intrasplenically grafted into nude mice. Thymidylate synthase (TS) and thymidine kinase (TK) mRNA expression, proliferation, apoptosis, and cell cycle alterations were studied. RESULTS: In vivo, 5-FU alone inhibited growth of only 3 of the 12 hCRCs tested and 3n-But alone had no effect; the 5-FU/3n-But combination inhibited growth of all 16 hCRCs tested. The hCRCs differed in their p53 and microsatellite instability status. 5-FU/3n-But decreased TK and TS mRNA expression by 20- and 40-fold, respectively, and TS activity by 75%, stopped cell proliferation without affecting cell differentiation, and significantly enhanced apoptosis. 3n-But potentiated the efficacy of Tomudex and methotrexate, 2 TS inhibitors, but not that of oxaliplatin. In vitro, 5-FU/3n-But inhibited [3H]thymidine but not bromodeoxyuridine incorporation and induced apoptosis in hCRC cell lines. Cells treated with 5-FU/3n-But did not accumulate in G1 nor in S phase of the cell cycle, while 5-FU and 3n-But arrested the cycle in S and in G1 phase, respectively. 3n-But prevented the cell rescue from 5-FU-induced cytotoxicity by uridine or thymidine. CONCLUSIONS: 3n-But and TS inhibitors acted synergistically against colorectal cancers, independently of the genetic alterations of the hCRCs. The mechanism of action of 5-FU/3n-But could be enhanced reduction of TS and prevention of thymidine salvage in DNA synthesis.

Clinical and experimental progression of a new model of human prostate cancer and therapeutic approach.

We report the clinical evolution of a prostate cancer, metastasizing to lungs and bones, recurring locally, and escaping from anti-androgen therapy. Key event of biological progression of the patient's tumor was the coincidence of allelic imbalance accumulation and of bone metastases occurrence. The recurrent tumor was established as the transplantable xenograft PAC120 in nude mice, where it grew locally. PAC120 displayed the same immunophenotype of the original tumor (positive for keratin, vimentin, prostatic acid phosphatase, and Leu-7) and expressed human HOXB9, HOXA4, HER-2/neu, and prostate-specific antigen genes, as detected by reverse transcriptase-polymerase chain reaction. It formed lung micrometastases detected by mRNA expression of human genes. Cytogenetic analysis demonstrated numerous alterations reflecting the tumor evolution. PAC120 was still hormone-dependent; its growth was strongly inhibited by the new gonadotropin-releasing hormone antagonist FE 200486 but weakly by gonadotropin-releasing hormone superagonist D-Trp(6)-luteinizing-hormone releasing hormone (decapeptyl). Tumor growth inhibition induced by anti-hormone therapy was linked to the hormone deprivation degree, more important and more stable with FE 200486 than with D-Trp(6)-luteinizing-hormone releasing hormone. Surgical castration of mice led to tumor regressions but did not prevent late recurrences. Transition to hormone-independent tumors was frequently associated with a mucoid differentiation or with a neuroendocrine-like pattern. Independent variations of mRNA expression of HER-2/neu and prostate-specific antigen were observed in hormone-independent tumors whereas HOXB9 gene expression was constant. In conclusion, PAC120 xenograft, a new model of hormone-dependent prostate cancer retained the progression potential of the original tumor, opening the opportunity to study the hormone dependence escape mechanism.