Biomedical subjects

Rolf Lewensohn

Publications and source records attributed to Rolf Lewensohn.

32 records · Page 2Linked to original sources

PubMed2003

Defective stress kinase and Bak activation in response to ionizing radiation but not cisplatin in a non-small cell lung carcinoma cell line.

We have here examined ionizing radiation (IR)-induced apoptotic signaling in one IR-sensitive small cell lung carcinoma (SCLC) and one resistant non-small cell lung carcinoma (NSCLC) cell line, both harboring mutant p53. In the sensitive SCLC cell line, IR induced conformational modulation of Bak and Bax, mitochondrial depolarization, and nuclear fragmentation. These events were not observed in the IR-resistant NSCLC cell line. However, in the same cells, cisplatin, a DNA-damaging drug, induced Bak and Bax modulation, mitochondrial depolarization, and nuclear fragmentation. Pre-mitochondrial signaling events were examined in order to further characterize the differing IR response. In the SCLC cell line, IR-induced apoptotic signaling was found to involve a MEKK1-related pathway and activation of the stress-activated kinases JNK and p38. In comparison, the NSCLC cell line had higher basal levels of activity of JNK and p38, and IR treatment did not further activate these kinases. However, NSCLC cells were sensitive to Bak modulation and apoptosis induced by a kinase-active mutant of MEKK1. Together, the results delineate a mechanism of IR resistance in NSCLC cells and indicate that IR and cisplatin induce Bak modulation and apoptosis via different pathways.

Apoptosis↗

PubMed2003

Expression of Ku86 confers favorable outcome of tonsillar carcinoma treated with radiotherapy.

BACKGROUND: To determine possible molecular markers for predicting radiosensitivity in squamous cell carcinoma, we have examined the relationship between pretreatment expression of the DNA damage recognition complex DNA-PK, its in vitro substrates, p53 and MDM2, local tumor control after radiotherapy (RT), and patient survival. METHODS AND MATERIALS: Formalin-fixed tumor biopsy specimens from 79 previously untreated patients with tonsillar carcinoma were analyzed by immunohistochemical methods. RESULTS: Tumors expressing high levels of Ku86 had better locoregional control in contrast to tumors expressing low levels of Ku86 (p =.023). Survival of patients with tumors expressing high levels of DNA-PKcs was significantly better than survival of patients with tumors expressing low levels of DNA-PKcs (p =.0024). p53 and MDM2 status alone did not correlate with survival of patients. However, patients with p53 tumors and high DNA-PKcs expression had significantly better survival than patients with p53+ tumors expressing low levels of DNA-PKcs (p =.0018). Furthermore, survival of patients with high expression of DNA-PKcs or Ku86 and low MDM2 levels was significantly better when compared with survival of patients with low DNA-PKcs or Ku86 and high MDM2 (p =.0017 and p =.0034, respectively). CONCLUSIONS: High expression of DNA-PKcs/Ku86 in combination with p53 negativity in tonsillar carcinoma correlates with better survival of patients. Identifying tumors with a phenotype predicting poor survival may be used to optimize treatment of patients with radioresistant tumors.

Aged↗

PubMed2003

The role of radiotherapy in treatment of stage I non-small cell lung cancer.

Most information on results with radiotherapy (RT) for stage I non-small cell lung cancer (NSCLC) is based on retrospective studies on RT-treated inoperable NSCLC cases. Thus, the role of RT for stage I NSCLC, as a curative modality, has not yet been established. A literature search for studies on stage I non-small cell lung carcinoma (NSCLC) treated by RT alone resulted in 18 papers published between 1988 and 2000. The majority of stage I patients received RT treatment because they were medically inoperable. The main contraindications for surgery were grave impairment of pulmonary function and serious cardiovascular disease. Local recurrence was the most common reason for treatment failure (median value 40%) but varied highly between the studies, ranging from 6.4 to 70%. In contrast with local recurrence, regional failure was not a major problem (0-3.2%). Generally, smaller tumour size, low T-stage and increased dose had a favourable impact on local control and increased local control was followed by increased survival. No serious treatment complications were recorded in the majority of these studies. Overall treatment results were, however, disappointing. The median survival in these studies ranged from 18 to 33 months. The 3- and 5-year overall survival was 34+/-9 and 21+/-8% (mean value+/-1 S.E.), respectively. The cause-specific survival at 3 and 5 years was 39+/-10 and 25+/-9% (mean value+/-1 S.E.), respectively. Dose escalation, in a setting with conformal RT using involved field or stereotactic RT, should be the focus of developmental therapeutic strategies with inoperable stage I NSCLC to improve local control and survival.

Adult↗

PubMed2003

Activity of hydrolytic enzymes in tumour cells is a determinant for anti-tumour efficacy of the melphalan containing prodrug J1.

Recently, we presented a series of melphalan containing di- and tripeptides with high cytotoxic activity and J1 (l-melphalanyl-p-l-fluorophenylalanine ethyl ester) was identified as one of the most interesting compounds. It was speculated that the increased activity compared to melphalan itself, demonstrated both in vitro and in vivo, resided in increased transport over the tumour cell membrane and/or hydrolytic cleavage and liberation of melphalan inside the cells. Indeed, overexpression of hydrolytic enzymes like peptidases, esterases and proteases has been described in several types of human malignancies, thus providing a target for selective chemotherapy. In this work, the details of the increased activity was further investigated and potential tumour selectivity is discussed. The intracellular delivery of melphalan is investigated in detail using peptidase resistant dipeptide derivatives, by enzyme inhibitors and probes for enzymatic activity and by studying the time dependency of drug effect as well as intracellular drug concentrations (cellular pharmacokinetics). The results show that the activity of the dipeptide mustards is highly dependent on intracellular hydrolysis, which result in rapid intracellular release of the alkylating unit (i.e. free melphalan) in cells with high enzymatic activity. The maximum intracellular melphalan concentration following J1 exposure was reached already after 15 min, thereafter declining with a half-life of approximately 1 h. This rapid intracellular loading resulted in less reduction of activity for J1 than for melphalan and six other standard drugs when human tumour cell lines were exposed to the drugs for a limited time (simulating short half-life in vivo). Peptidase inhibitors inhibited the activity and intracellular release of melphalan, and dipeptide derivatives designed to resist the action of peptidases was less active than the corresponding normal dipeptide.

Antineoplastic Agents, Alkylating↗

PubMed2003

Antitumor activity of the alkylating oligopeptides J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester) and P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester): comparison with melphalan.

Peptichemio, a mixture of six short oligopeptides all comprising the alkylating amino acid m-L-sarcolysin, has shown clinical activity in several malignancies. Previous studies have suggested that activity mainly resides in one of the peptides, P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester). In the present study the in vitro activity of P2 was further investigated and compared to melphalan and the novel alkylating dipeptide J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester), which is structurally related to P2 and melphalan. Cytotoxic activity was studied using patient tumor cells in a non-clonogenic cytotoxicity assay, whereas cellular response, and kinetics thereof, were studied in the lymphoma cell line U-937 GTB. Cellular metabolism was studied using microphysiometry, kinetic effects on macromolecular synthesis by radiolabeled substrate incorporation and, finally, the microculture kinetic assay of apoptosis was used to monitor morphologic changes following drug exposure. The assays compared P2 favorably with melphalan. Interestingly J1 was even more cytotoxic, and produced more pronounced effects in the kinetic assays for macromolecular synthesis, metabolic activity and apoptosis. The results indicate that the delivery properties of J1 are improved compared to those of melphalan and P2.

Antineoplastic Agents, Alkylating↗

PubMed2003

X-ray-induced DNA double-strand breaks in mouse l1210 cells: a new computational method for analyzing neutral filter elution data.

The aim of this article is to present a method for studying the shape of the dose and repair responses for X-ray-induced double-strand breaks (DSBs) as measured by neutral filter elution (NFE). The approach is closely related to a method we developed for the use of specific molecular size markers and used for determination of the absolute number of randomly distributed radiation-induced DSBs by pulsed-field gel electrophoresis (PFGE). Mouse leukemia L1210 cells were X-irradiated with 0-50 Gy. Samples were then evaluated both with PFGE and with NFE. Assuming that with both migration (PFGE) and elution (NFE), a heterogeneous population of double-stranded DNA fragments will start with the smallest fragments and proceed with increasingly larger fragments, it is possible to match the migration behavior of fractions of fragments smaller than a certain size to the fraction eluted at a specific time. This assumption does not exclude the possibility of DNA being sheared in the NFE filter. The yield, as determined by the size markers in PFGE, was used to find the corresponding elution times in the NFE experiment. These experimentally used elution times could then reversely be interpreted as size markers which finally were used to calculate DSBs/Mbp as a function of X-ray dose. The resulting lines were almost straight. The data were also plotted as relative elution and showed that, as expected, the dose response then appears with a more pronounced sigmoid shape.

Animals↗

PubMed2003

Structure-activity relationship for alkylating dipeptide nitrogen mustard derivatives.

The strategy of using small peptides for effective targeting of tumor cells in chemotherapy has proven beneficial. Recently we showed that J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester), an alkylating nitrogen mustard-containing dipeptide, exhibited strong cytotoxic activity in fresh human tumor samples in addition to rapid and pronounced inhibition of macromolecular syntheses and cellular respiration in the human tumor lymphoma cell line U-937 GTB. In this study, an additional series of 17 nitrogen mustard-containing dipeptides has been synthesized and analyzed for cytotoxic activity in a panel of 10 human tumor cell lines. The results were compared to the single amino acid mustard derivative melphalan and its ethyl and isopropyl esters. Also P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester), a tripeptide that previously has shown impressive effects in human tumor cells, was used as reference. The tested compounds displayed various activities in the different cell lines but also showed a high correlation, indicating a similar mechanism of action. Factors like amino acid composition, amino acid sequence, modifications of the C- and N-termini, and to a minor extent the lipophilicity of the dipeptide derivatives appear to influence the in vitro activity. The results indicate that the activity of these compounds not only relies on their chemical reactivity, but also on active biological interactions such as transport across membranes and/or enzymatic liberation of reactive molecular entities.

Alkylation↗

PubMed2003

Antitumor activity of the novel melphalan containing tripeptide J3 (L-prolyl-L-melphalanyl-p-L-fluorophenylalanine ethyl ester): comparison with its m-L-sarcolysin analogue P2.

Peptichemio (PTC), a mixture of six oligopeptides all containing m-L-sarcolysin, has previously shown impressive results in clinical trials. The tripeptide P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester) has been suggested as the main contributor to PTC activity. In contrast to its analogue melphalan, m-L-sarcolysin never reached clinical use. To allow a direct comparison, the corresponding melphalan containing tripeptide J3 (L-prolyl-L-melphalanyl-p-L-fluorophenylalanine ethyl ester) was synthesized and its activity was compared with that of P2; the activities of melphalan and m-L-sarcolysin were studied in parallel. Cytotoxic activity in human tumor cell lines and some fresh human tumor specimens were analyzed as well as effects on cellular metabolism, macromolecular synthesis, and preliminary evaluation of the cell death characteristics. The results show that melphalan and m-L-sarcolysin display similar activity in these systems and that the tripeptides were more active than their parent monomers. Surprisingly however, the melphalan containing tripeptide J3 demonstrated a significantly more rapid and stronger activity than the m-L-sarcolysin analogue P2. Finally, the in vivo toxicity and activity of melphalan and J3 were investigated in mice bearing human leukemia cells in s.c. fibers. The in vitro results seem translatable into the in vivo situation, demonstrating better antileukemic effect of J3 but similar side effects as melphalan.

Antineoplastic Agents, Alkylating↗

PubMed2002

Expression of inhibitor of apoptosis proteins in small- and non-small-cell lung carcinoma cells.

Small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cells both initiate apoptotic signaling, resulting in caspase activation, after treatment with anti-cancer agents. However, in contrast to SCLC cells, NSCLC cells do not fully execute apoptosis. The apoptotic process in NSCLC cells seems to be blocked downstream of caspase activation, thus the failure of NSCLC cells to execute apoptosis could result from inhibition of active caspases by inhibitor of apoptosis proteins (IAPs). Here we investigate the mRNA and protein expression of IAPs in a panel of SCLC and NSCLC cell lines. The NSCLC cell lines had a stronger cIAP-2 expression at both mRNA and protein levels, while the SCLC cell lines had a higher level of XIAP protein. Expression of cIAP-1, cIAP-2, and XIAP, the most potent caspase inhibitors, was further investigated in three lung carcinoma cell lines after treatment with 8 Gy of ionizing radiation or etoposide (VP16). In response to treatment, the level of IAPs was not altered in a way that explained the differences in cellular chemo- and radiosensitivity. The intracellular localization of IAPs was analyzed in untreated and treated lung cancer cells. Surprisingly, we found that cIAP-2 was mainly detected in the mitochondrial fraction, although the function of this protein in mitochondria is unknown. No major relocalization of IAPs was observed after treatment. Taken together, these results indicate that IAPs alone are not the main factor responsible for the resistance of NSCLC cells to treatment.

Antineoplastic Agents, Phytogenic↗

PubMed2002

Synthesis and cytotoxicity of the dihydrated complex of oxaliplatin.

A new way of synthesizing the dihydrated oxaliplatin complex (DOC) is presented and its cytotoxicity is compared to that of oxaliplatin and cisplatin. By hydrolyzing oxaliplatin in aqueous sodium hydroxide at 70 degrees C, DOC was formed in less than 1 h. Cytotoxicity was studied in the non-small cell lung cancer cell line A549 using the fluorescent microculture cytotoxic assay. Oxaliplatin and cisplatin had similar cytotoxicity profiles, whereas DOC was found to be considerably more toxic. The cytotoxicity of oxaliplatin might, at least in part, be mediated through the formation of DOC.

Antineoplastic Agents↗

PubMed2002

[Management of radiation injuries--new organization, new guidelines].

The medical management of radiation accident victims is based on a rapid assessment and reconstruction of the radiation dose and quality combined with prompt, targeted treatment efforts performed at specialized medical units. To facilitate such medical emergency preparedness in Sweden, the National Board of Health and Welfare has recently established a new Centre for Radiation Medicine, located at the Karolinska institute in Stockholm. Among the tasks of this centre are also to contribute with health care information, education, advice and carry out research activities in areas related to medical effects of ionizing radiation. A close collaboration is being established with the Swedish Radiation Protection Authority and also with various other national and international bodies. The initiation of the Centre for Radiation Medicine comes in parallel with a rapid development of the understanding of and treatment options for patients acquiring an acute radiation syndrome, mainly due to improved diagnostic procedures combined with novel therapeutic options, such as stem cell transplantation and cytokine administration.

Academies and Institutes↗

PubMed2002

Mitochondrial dysfunction is an essential step for killing of non-small cell lung carcinomas resistant to conventional treatment.

Apoptosis, a tightly controlled multi-step mechanism of cell death, is important for anti-cancer therapy-based elimination of tumor cells. However, this process is not always efficient. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) cells display different susceptibility to undergo apoptosis induced by anticancer treatment. In contrast to SCLC, NSCLC cells are cross-resistant to a broad spectrum of apoptotic stimuli, including receptor stimulation, cytotoxic drugs and gamma-radiation. Since resistance of tumor cells to treatment often accounts for the failure of traditional forms of cancer therapy, in the present study attempts to find a potent broad-range apoptosis inductor, which can kill therapy-resistant NSCLC cells were undertaken and the mechanism of apoptosis induction by this drug was investigated in detail. We found that staurosporine (STS) had cell killing effect on both types of lung carcinomas. Release of cytochrome c, activation of apical and effector caspases followed by cleavage of their nuclear substrates and morphological changes specific for apoptosis were observed in STS-treated cells. In contrast to treatment with radiation or chemotherapy drugs, STS induces mitochondrial dysfunction followed by translocation of AIF into the nuclei. These events preceded the activation of nuclear apoptosis. Thus, in lung carcinomas two cell death pathways, caspase-dependent and caspase-independent, coexist. In NSCLC cells, where the caspase-dependent pathway is less efficient, the triggering of an AIF-mediated caspase-independent mechanism circumvents the resistance of these cells to treatment.

Amino Acid Chloromethyl Ketones↗

PubMedDate not supplied

Radiation-induced cell cycle response in lymphocytes is not related to clinical side-effects in breast cancer patients.

BACKGROUND: We examined whether development of radiation-induced lung injury after irradiation for breast cancer correlates with lymphocyte radiosensitivity (LRS) in vitro. MATERIALS AND METHODS: Patients were selected from a cohort of 177 patients, who were treated with adjuvant postoperative radiotherapy (RT) for loco-regional breast cancer, and included 14 patients who had severe early lung injury measured as respiratory symptoms caused by RT and treated with corticosteroids (i.e. "cases") and a corresponding 14 control patients without such symptoms. LRS was measured as the postirradiation fraction of mitogen-stimulated blood lymphocytes in S- and G2-phase. Plasma levels of TGF-beta 1 and thiols, both suggested to be involved in the pathogenesis of radiation-induced lung injury, were also analysed. RESULTS: The result showed that cells from the controls responded to a higher extent to mitogen stimulation than cells from the cases (p < 0.05). Analysis of the fraction of S- and G2-phase cells after irradiation showed no significant difference between the two groups (p = 0.57 and 0.31, respectively). There was no difference in plasma levels of TGF-beta 1 and thiols in patients who did or did not develop pulmonary injury after RT. Tamoxifen administered before or at RT did not influence the incidence of pulmonary reactions. CONCLUSION: No differences in LRS were found between breast cancer patients with lung complications after RT and matched control patients without complications.

Adult↗

PubMedDate not supplied

Heat shock protein 72 does not modulate ionizing radiation-induced apoptosis in U1810 non-small cell lung carcinoma cells.

Heat shock proteins (HSP) have been shown to interfere with apoptosis signaling, suggesting that there might be a role for these proteins as mediators of resistance to ionizing radiation (IR)-induced apoptosis. Protein expression of the stress inducible heat shock proteins, HSP72 and HSP27, was analyzed in a panel of lung carcinoma cell lines displaying various degrees of radiosensitivity. Expression of HSP72 was high in all cell lines investigated while HSP27 was present in all non-small cell lung carcinoma (NSCLC) and 6/9 small cell lung carcinoma (SCLC) cell lines. Heat shock, but not IR, induced or further increased the expression of HSP27 and HSP72. Moreover, elevation of heat shock protein level prior to irradiation did not attenuate IR-induced apoptotic signaling or the induction of apoptosis. Protein level of HSP72 was downregulated in a radioresistant NSCLC cell line by RNA interference. However, this did not sensitize cells to treatment with DNA-damaging agents such as IR, cisplatin or VP16. Thus, the results from this first study on the relationship between stress-inducible HSP expression and IR-induced apoptosis in lung cancer cells do not support a role for HSP 27 and 72 in the radioresistance of NSCLC cells.

Apoptosis↗