The effect of L-2-amino-4-methoxy-trans-3-butenoic acid on serine hydroxymethyl transferase

The tumour growth inhibitor L-2-amino-4-methoxy-trans-3-butenoic acid (Ro07-7957) inhibits serine hydroxymethyltransferase in cytosolic extracts of Walker carcinoma non-competitively with respect to L-serine with an apparent inhibition constant similar to the K_m-value for L-serine. The kinetics of inactivation suggest that it reacts as an irreversible substrate analogue. Incubation of Walker cells with Ro07-7957 causes an increase in serine hydroxymethyltransferase activity which is most pronounced at concentration ≤LD₅₀. This increase in enzyme activity does not occur in the presence of cycloheximide. These results suggest that inhibition of serine hydroxymethyltransferase in intact cells is accompanied by an increase in enzyme biosynthesis and that the growth inhibitory property of Ro07-7957 does not involve interference with the conversion of serine to glycine.     

Application of Fluorescence Microscopy to Measure Apoptosis in Jurkat T Cells After Treatment with a New Investigational Anticancer Agent (N.C.1213)

1. Apoptosis as the mechanism of cell death induced by a new cytotoxic and anticancer agent (N.C.1213) was investigated by morphological and biochemical criteria in human Jurkat T leukemia cells.2. The effect of N.C.1213 on the survival of Jurkat T, LV-50, H-9, and Molt-3 cells was measured. Jurkat T cells exhibited the highest response, with less than 10% of the cells remaining viable after exposure to 10 M N.C.1213 for a 24 hr period. All other cell cultures were also affected but to a lesser extent.3. With the use of a fluorescence microscope, several morphological features characteristic of apoptosis such as condensed chromatin and apoptotic bodies were indemnified in Jurkat T cells after exposure to N.C.1213 and melphalan. The results indicated that melphalan was more cytotoxic than N.C.1213 as shown by the dye exclusion test. However, N.C.1213 showed a greater apoptotic index than melphalan. The IC₅₀ of N.C.1213 in Jurkat T cells was determined to be 3.5 M 4. A DNA ladder (fragmentation of DNA into multimers of approximately 200 base pairs), which is one characteristic feature of apoptosis, was not detected when Jurkat T cells were exposed to N.C.1213. Hence it is probable that the key morphological events in apoptosis observed in the present experimental conditions precede the internucleosomalcleavage of DNA.     

DNA repair pathways in human multiple myeloma

Every day, cells are faced with thousands of DNA lesions, which have to be repaired to preserve cell survival and function. DNA repair is more or less accurate and could result in genomic instability and cancer. We review here the current knowledge of the links between molecular features, treatment, and DNA repair in multiple myeloma (MM), a disease characterized by the accumulation of malignant plasma cells producing a monoclonal immunoglobulin. Genetic instability and abnormalities are two hallmarks of MM cells and aberrant DNA repair pathways are involved in disease onset, primary translocations in MM cells, and MM progression. Two major drugs currently used to treat MM, the alkylating agent Melphalan and the proteasome inhibitor Bortezomib act directly on DNA repair pathways, which are involved in response to treatment and resistance. A better knowledge of DNA repair pathways in MM could help to target them, thus improving disease treatment.     

Effect of hyperthermia and chemotherapeutic agents on TRAIL-induced cell death in human colon cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In the previous study [Yoo and Lee, 2007], we have reported that hyperthermia could enhance the cytotoxicity of TRAIL-induced apoptosis. We observed in human colorectal cancer cell line CX-1 that TRAIL-induced apoptotic death and also that mild hyperthermia promoted TRAIL-induced apoptotic death through caspase activation and cytochrome-c release. Although its effects in vivo are not clear, hyperthermia has been used as an adjunctive therapy for cancer. Hyperthermia is often accompanied by chemotherapy to enhance its effect. In this study, CX-1 colorectal adenocarcinoma cells were treated with TRAIL concurrently with hyperthermia and oxaliplatin or melphalan. To evaluate the cell death effects on tumor cells via hyperthermia and TRAIL and chemotherapeutic agents, FACS analysis, DNA fragmentation, and immunoblottings for PARP-1 and several caspases and antiapoptotic proteins were performed. Activities of casapse-8, caspase-9, and caspase-3 were also measured in hyperthermic condition. Interestingly, when analyzed with Western blot, we detected little change in the intracellular levels of proteins related to apoptosis. Clonogenic assay shows, however, that chemotherapeutic agents will trigger cancer cell death, either apoptotic or non-apoptotic, more efficiently. We demonstrate here that CX-1 cells exposed to 42 degrees C and chemotherapeutic agents were sensitized and died by apoptotic and non-apoptotic cell death even in low concentration (10 ng/ml) of TRAIL.     

Reduced bone marrow stem cell pool and progenitor mobilisation in multiple myeloma after melphalan treatment

The content of stem cells was analysed in bone marrow samples from 75 multiple myeloma patients. In unstimulated bone marrow the percentage of CD34+cells was significantly reduced in 11 patients previously treated with melphalan-prednisolone (MP)(median= 0.15%) compared to median 0.87% in 31 untreated patients (P=0.0001). The bone marrow cellularity in the two groups did not differ. There was no correlation between the number of courses or total dose of melphalan and content of CD34+cells in the bone marrow. The clonogenicity as, well as the ability to expand the marrow stem cell pool during growth factor treatment were also reduced in MP treated patients compared to untreated patients. Analysis of different subsets of CD34+ cells revealed no influence on the pre B cell compartment in the bone marrow by MP treatment, but the committed stem cells (CD34+CD38+) were reduced more than the uncommitted stem cells (CD34+CD38—) in the MP treated group compared to the untreated patients. Mobilisation to and harvest of total number of CD34+ cells from peripheral blood was also reduced in the MP treated group. There was, however, no difference in the distribution between CD34+CD38+and CD34+CD38—populations in the leukapheresis products in the untreated and the melphalan-treated group, suggesting selective mobilisation of CD34+CD38+ cells and/or differentiation of CD34+ CD38-cells during growth factor stimulation. We conclude that melphalan decreased the number of stem cells in the bone marrow, the ability to expand the stem cell pool and mobilise stem cells to the pheripheral blood.     

Gene expression changes in melanoma metastases in response to high-dose chemotherapy during isolated limb perfusion

Despite recent advances in melanoma therapy, disseminated melanoma still lacks effective treatment, and recurrence of the tumor frequently occurs, even after high-dose chemotherapy. The mechanisms responsible for this chemoresistance or for the formation of new relapses remain poorly understood. Using a human 'model', in which the isolated limb is perfused with high doses of the chemotherapeutic melphalan (ILP), we identified a five-gene set (ATF3, CYR61, IER5, IL6, and PTGS2) of stress-induced genes that was consistently upregulated after ILP in all in-transit metastatic melanoma samples as well as in three melphalan-treated melanoma cell lines. Early post-ILP relapses retained these elevated expressions, whereas the expression of these genes returned to their original levels in late post-ILP recurrences. In addition, we identified upregulation of these genes in the A375 cell line's side population (SP) and melanospheres, established methods to enrich for candidate cancer stem cells (CSCs), which are considered chemoresistant and tumorigenic, and thus proposed to be responsible for tumor relapse. Our data identify an immediate and short-term upregulation of early stress-responsive genes that are potentially linked to chemoresistance and CSCs.     

Effects of amylobarbitone on the frequency of chromosomal aberrations in human lymphocytes determined by chlorambucil and melphalan in vitro

Evidence is produced that the amount of chromosomal (chromatid) damage in human lymphocytes exposed throughout culture to chlorambucil (CBC) and to melphalan (MELPH) is considerably reduced by the presence in these cultures also of amylobarbitone. It is suggested that this could be due to similar of production or activity of “hydroxylase” type enyzmes by the barbiturate and that these alkylating agents are therefore more rapidly degraded to derivatives which cause less chromosomes breakage than the “intact” drugs. Such enzymes have previously been considered to be active almost entirely in the smooth type of hepatic microsomes.     

Effects of alkylating agents on the DNA replication of cultured Yoshida sarcoma cells

Logarithmically growing Yoshida sarcoma cells were treated for 1 h with low (2 decades cell kill) or high (more than 6 decades cell kill) doses of alkylating agents. Pulse and chase labelled DNA from treated cells were studied by alkaline sucrose gradient centrifugation. Nitrogen mustard (HN-2), 4-hydroperoxycyclophosphamide (CY-OOH), melphalan (L-PAM) and chlorambucil (CA) had no effect on the elongation rate of newly replicated DNA, both at low and high doses, although per cell the rate of DNA synthesis declined as inferred from the rates of [³H] thymidine incorporation compared to the increase in numbers of S phase cells in the treated populations. It is concluded that these drugs act specifically on the initiation step of the DNA replication, leaving chain elongation undisturbed. At low doses the chemically related sulphur mustard (SM) had also no effect on the maturation of new DNA but at high doses a decreased elongation rate was observed. A transient inhibition of chain growth was observed following treatment with a low dose of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In contrast, the intercalating agent adriamycin showed a severe but delayed effect resulting in an almost complete block of the maturation.     

Synthesis,Molecular Docking and Preliminary Antileukemic Activity of 4-Methoxybenzyl Derivatives Bearing Imidazo[2,1-b][1,3,4]thiadiazole

In this study, we synthesized 22 compounds in a series with various substitution on imidazo[2,1-b][1,3,4]thiadiazole. The potential cytotoxic activity of these compounds investigated in leukemia cell lines by Differential Nuclear Staining (DNS). Our results identified two compounds, 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate and 6-(4-chlorophenyl)-2-(4-methoxybenzyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde, exhibited the most cytotoxic effect against murine leukemia cells (L1210), human T-lymphocyte cells (CEM) and human cervix carcinoma cells (HeLa) with IC₅₀ values ranging between 0.79 and 1.6 μM. The results indicate that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate is inducing phosphatidylserine externalization and caspase-3 activation which are both a hallmark of apoptosis. Docking studies showed that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate binds within the active sites of transforming growth factor beta (TGF-β) type I receptor kinase domain by strong hydrogen binding and hydrophobic interactions.     

Rationale for using TNFα and chemotherapy in regional therapy of melanoma

Recombinant tumor necrosis factor-alpha (rTNFα) has potent antitumor activity in experimental studies on human tumor xenografts. However, in humans, the administration of rTNFα is hampered by severe systemic side-effects. The maximum tolerated dose range from 350 to 500 mg/m², which is at least 10-fold less than the efficient dose in animals. Isolation perfusion of the limbs (ILP) allows the delivery of high dose rTNFα in a closed system with acceptable side-effects. A protocol with a triple-drug regimen was based on the reported synergism of rTNFα with chemotherapy, with interferon-y, and with hydperthermia. In melanoma-in-transit metastases (stage IIIA or AB) we obtained a 91% complete response, compared with 52% after ILP with melphalan alone. Release of nanograms levels of TNFα in the systemic circulation was evident but control of this leakage and appropriate intensive care resulted in acceptable toxicity. Angiographic, immunohistological, and immunological studies suggest that the efficacy of this prtocol is due to a dual targeting: rTNFα activates and electively lyses the tumor endothelial cells while melphalan is mainly cytoxic to the tumor cells. ILP with rTNFα appears to be a useful model for studying the biochemotherapy of cancer in man.