bcl-2 antisense therapy chemosensitizes human melanoma in SCID mice.

Malignant melanoma is a prime example of cancers that respond poorly to various treatment modalities including chemotherapy. A number of chemotherapeutic agents have been shown recently to act by inducing apoptosis, a type of cell death antagonized by the bcl-2 gene. Human melanoma expresses Bcl-2 in up to 90% of all cases. In the present study we demonstrate that bcl-2 antisense oligonucleotide treatment improves the chemosensitivity of human melanoma grown in severe combined immunodeficient (SCID) mice. Our findings suggest that reduction of Bcl-2 in melanoma, and possibly also in a variety of other tumors, may be a novel and rational approach to improve chemosensitivity and treatment outcome.

Activated N-ras contributes to the chemoresistance of human melanoma in severe combined immunodeficiency (SCID) mice by blocking apoptosis.

Activation of the N-ras gene by point mutations occurs in about 15 % of all human melanomas. Using recently established melanoma severe combined immunodeficiency-human mouse xenotransplantation models, here we further investigate the biological significance of these mutations. We demonstrate that activated N-ras significantly contributes to the chemoresistance of human melanoma both in vitro and in vivo by blocking apoptosis. Overexpression of wild-type N-ras had no such effects. With antisense oligonucleotides and farnesyltransferase inhibitors, tools capable of blocking Ras function on the therapeutic horizon, our observation that activated N-ras is not a bystander but a factor worth targeting to improve therapeutic outcome in melanoma gains additional importance.

TP53 mutation and p53 overexpression for prediction of response to neoadjuvant treatment in breast cancer patients.

The value of p53 to predict the cytotoxic effect of two commonly used chemotherapy regimens was assessed in patients with advanced breast cancer. Response to a DNA-damaging combination therapy [fluorouracil, epirubicin, cyclophosphamide (FEC] considered to induce p53-dependent apoptosis was compared with a microtubule stabilizing therapy (paclitaxel) expected to be independent of p53 function. The p53 status of the patients' breast tumors was assessed using both immunohistochemistry (IHC) and direct sequencing of the entire p53 gene. p53 findings were correlated with treatment response, and linkage between p53 function and cellular response was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. In a series of 67 breast tumors, 19% had TP53 gene mutations, 40% had a positive p53 IHC, and 12% had both. In the FEC group, treatment failure was related to both the presence of TP53 gene mutations (P = 0.0029) and a positive IHC (P < 0.0001). Apoptosis was almost exclusively found in tumors having normal p53 in both parameters (P < 0.0001). In the paclitaxel group, treatment response was neither related to apoptosis nor to normal p53. Combination of sequencing and IHC results revealed a significant association between abnormal p53 and response to paclitaxel (P = 0.011). We found TP53 mutations, as well as p53 protein overexpression, to be associated with response to chemotherapy. Whereas clinical response to FEC was found to be dependent on normal p53, the cytotoxicity of paclitaxel was related to defective p53. The efficiency of paclitaxel during mitosis might be supported by lack of G1 arrest due to p53 deficiency. Therefore, patients with p53-deficient tumors may benefit from paclitaxel.

Farnesylthiosalicylic acid inhibits the growth of human Merkel cell carcinoma in SCID mice.

Merkel cell carcinoma (MCC) is a neuroendocrine malignancy showing poor response to a variety of therapeutic strategies. We evaluated the antitumor activity of S-trans, trans-farnesylthiosalicylic acid (FTS), a new inhibitor of Ras signal transduction, in a newly established SCID mouse xenotransplantation model for human MCC (seven animals per group). FTS injected intraperitoneally at 5 mg/kg per day for 2 weeks up-regulated the tumor suppressor p53 and induced tumor cell apoptosis in established MCCs growing subcutaneously in SCID mice. These effects led to a statistically significant inhibition of MCC growth (P<0.002). The mean tumor weights following FTS or control treatment were 0.32+/-0.15 g and 1.08+/-0.29 g, respectively. There was no evidence of FTS related toxicity at the effective dose used. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for MCC and possibly for other tumors that rely on tyrosine kinase signaling.

Bcl-2 antisense oligonucleotides (G3139) inhibit Merkel cell carcinoma growth in SCID mice.

Merkel cell carcinoma was first described in 1972 by Toker and is an aggressive neuroendocrine skin tumor with a high metastatic potential. Merkel cell carcinoma is thought to derive from the neuroendocrine (Merkel) cells of the skin, although in contrast to fetal and especially adult Merkel cells, Merkel cell carcinomas express high levels of the Bcl-2 oncoprotein. Bcl-2 is capable of blocking programmed cell death and has been shown to play an important role in normal cell turnover, tumor biology, and chemoresistance. High Bcl-2 expression leading to prolonged survival of cells may therefore be of importance in the biological and clinical characteristics of Merkel cell carcinoma. In a SCID mouse xenotransplantation model for human Merkel cell carcinoma, we investigated the influence of the bcl-2 antisense oligonucleotide G3139 (Genta) on tumor growth in comparison with control oligonucleotides or cisplatin. Bcl-2 antisense treatment, targeting the first six codons of the bcl-2 mRNA, resulted in either a dramatic reduction of tumor growth or complete remission, whereas reverse sequence and two-base mismatch control oligonucleotides or cisplatin had no significant antitumor effects compared with saline-treated controls. Apoptosis was enhanced 2.4-fold in the bcl-2 antisense treated tumors compared with the saline-treated group, and no other treatment showed a comparable increase in apoptosis. Our findings suggest that bcl-2 antisense treatment may be a novel approach to improve treatment outcome of human Merkel cell carcinoma.

Influence of increased c-Myc expression on the growth characteristics of human melanoma.

Overexpression of the proto-oncogene c-myc has been associated with neoplastic transformation in a variety of tumors. For human melanoma high c-myc expression has been found in the vertical growth phase and higher positivity reported in metastases than primary tumors. The principle aim of this study was to determine, whether c-Myc expression influences the metastatic behavior of human melanoma in the absence of lymphocyte-mediated immune phenomena. The growth characteristics and tumor biology of two c-myc transfectants of the human melanoma cell line IGR39D, expressing c-Myc 1.7 and three times over baseline and the respective vector control were analyzed both in vitro and in a severe combined immunodeficient mouse model in vivo. Both c-myc transfectants showed increased growth rates, anchorage independent growth and directed cell movement in culture. Subcutaneously implanted IGR39D melanomas highly overexpressing c-Myc spontaneously formed macroscopic metastases (lymph nodes and lung) in severe combined immunodeficient mice in all cases (n = 7 per group), whereas less prominent c-Myc overexpression caused the development of only lung micrometastases. During the time period leading to terminal disease in animals injected with c-myc transfected human melanoma cells, melanoma development was not seen in vector controls. These findings suggest that constitutive high c-Myc expression in human melanoma results in a more aggressive growth behavior both in vitro and in vivo and favors metastasis in severe combined immunodeficient mice by factors unrelated to immune phenomena such as class I human leukocyte antigen downregulation known to be associated with c-Myc expression.

Perilesional injection of r-GM-CSF in patients with cutaneous melanoma metastases.

Based on evidence that granulocyte-macrophage colony stimulating factor (GM-CSF) induces a potent systemic antitumor immunity, we tested recombinant GM-CSF in advanced melanoma. Seven patients with histologically confirmed cutaneous melanoma metastases were treated with perilesional intracutaneous injections of recombinant GM-CSF and observed for a follow-up time of 5 y. All but two patients had a decrease in the total number of metastases. At the end of the 5 y follow-up three of the seven patients are still alive with only one patient receiving other than surgical therapy, and one patient died tumor free at the age of 93. The remaining three patients died from progressive melanoma. Perilesional intradermal GM-CSF therapy resulted in a mean survival time of 33 mo. The treatment was well tolerated and no side-effects other than local erythema at the injection sites and mild drowsiness were seen. Immunohistochemical analysis with staining for CD14 and GM-CSF receptor demonstrated an increased infiltration of monocytes into both injected and noninjected cutaneous melanoma metastases compared with lesions excised prior to the initiation of therapy. The same was true for CD4- and CD8-positive lymphocytes. This phenomenon, together with GM-CSF-induced leukocyte counts of more than 20,000 during therapy, support the possible impact of a systemic over a locally induced reaction by GM-CSF. To our knowledge this is the first report that intracutaneously injected GM-CSF results in long-lasting reduction of melanoma metastases.

Mutated N-ras upregulates Bcl-2 in human melanoma in vitro and in SCID mice.

Activation of the N-ras gene by point mutation occurs in about 15% of all human melanomas. In recently established severe combined immunodeficiency (SCID) mouse xenotransplantation models for human melanoma, we demonstrated that mutated N-ras not only contributes to tumour growth by enhancing cellular proliferation, but also by blocking apoptosis. Mutated N-ras overexpression protected human melanomas from naturally occurring apoptosis and, in a more pronounced way, from chemotherapy-induced apoptosis in vitro and in vivo. Given the potential clinical importance of these findings we sought to determine the underlying mechanism. We found that mutated N-ras specifically upregulates the expression of the anti-apoptosis gene bcl-2 in two human melanoma cell lines in vitro and in SCID mice. Neither the expression of the anti-apoptotic protein Bcl-xL nor that of the pro-apoptotic proteins Bax and Bak were altered in cells expressing mutated N-Ras. The increase in Bcl-2 expression mediated by mutated ras therefore qualifies as a rational explanation for the enhanced chemoresistance of human melanoma expressing mutated N-Ras.

Expression of Bcl-2 family members in human melanocytes, in melanoma metastases and in melanoma cell lines.

In human melanoma no complete information about the expression of the apoptosis-promoting and apoptosis-inhibiting members of the Bcl-2 family has been available to date. In this study we have investigated by Western blotting the expression pattern of Bcl-2 and its homologues Bax, Bak, Bcl-xL, Bcl-xS, Mcl-1 and Bad in 12 distant lymph node metastases from patients who have been treated by different regimes, in nine newly established cell lines of these metastases, in three cell lines obtained from other sources and in primary melanocytic cell lines from three neonatal and two adult subjects. Taken together, our data suggest that Bax, Bak, Bad, Bcl-xL and Mcl-1 are expressed in addition to Bcl-2 in both normal melanocytes and in cell lines established from melanoma metastases. Regarding the role of Bcl-2 and its homologues, our data suggest that expression of this class of proteins is widespread and qualitatively similar in melanoma cell lines and normal human melanocytes. Although the expression of these proteins might affect growth behaviour and the progression of melanomas, our results are not compatible with the hypothesis that the Bcl-2 homologues investigated play a dominant role in the process of malignant transformation of melanocytes.

Erythropoietin receptor expression in human melanoma cells.

Erythropoietin is well known for its role in the control of erythropoiesis, where it acts by binding to its cognate receptor (EpoR) on the surface of erythroid progenitor cells. Here we present the novel finding that the EpoR is also expressed in cells of the melanocytic lineage. It is expressed in transformed cell lines established from normal melanocytes and also in established human melanoma cell lines derived from melanoma metastases, but not in normal primary human melanocytes. The analysis of individual subclones isolated from spontaneously transformed melanocytes revealed that approximately 50% of all the clones examined expressed the EpoR. Further analysis of the individual growth characteristics of EpoR-positive and EpoR-negative clones indicated that, under standard cell culture conditions, expression of the receptor did not affect cell growth. Expression of this receptor is consequently most likely driven by an event that is associated with, but not absolutely required for, the transformed phenotype. While the definite function of this receptor in melanoma cells is still unknown and additional studies are required, our findings support the hypothesis that the EpoR may serve as a progression marker for human melanoma. This observation might be useful in the early diagnosis of melanoma.

Elevated procaspase levels in human melanoma.

In this study procaspase expression levels were investigated by Western blotting in a panel of established melanoma cell lines, transformed melanocytic cell lines and normal primary melanocytes. Upstream caspases such as procaspase-8 that contain a death effector domain were found to be overexpressed in transformed melanocytes and melanoma cell lines compared with melanocytes. Heterogeneous levels of procaspase-8 were seen in melanoma cells, including one cell line that completely lacked procaspase-8 expression. Procaspase-10 is generally overexpressed in transformed melanocytes and melanoma cell lines. Expression of the downstream procaspases-3 and -7 was increased in melanoma cells compared with normal melanocytes. Procaspases containing caspase recruitment domains such as procaspase-2 were expressed at similar levels in nearly all the cell lines investigated. Reduced levels of procaspase-1 compared with normal melanocytes were detected in transformed melanocytes and melanoma cell lines. These data indicate that procaspase levels in general increase during the malignant transformation of melanocytic cells.

Technetium-99m-tetrofosmin: a new agent for melanoma imaging?

The aim of our study was to examine whether technetium-99m 1,2-bis[bis(2-ethoxyethyl) phosphino]ethane (tetrofosmin) a lipophilic, cationic tracer which was first developed for myocardial perfusion imaging, could be a new radiopharmaceutical for melanoma imaging. We therefore used two human cell lines, SK-MEL 28 and 5i8 A2 (n = 6, cell concentration 106/ml, incubation at 22 degrees C and 37 degrees C, 50-100 approximately lCi/ml Tc-99m-tetrofosmin, time of incubation 10-180 minutes). The cellular uptake by both cell lines was determined. In contrast to another non- melanoma tumor cell line MCF-7 (a human adenocarcinoma breast cancer) which reached steadystate almost immediately (within 10 minutes), the cellular uptake of SK-MEL-28 increased after 60 minutes and showed a very high uptake (> 10%) after 120 minutes and decreased after 180 minutes (6-8%), while the uptake in 518 A2 cells was about 5% after 90 minutes. Our data show that Tc-99m-tetrofosmin could be a promising agent for melanoma imaging.

A novel Ras antagonist regulates both oncogenic Ras and the tumor suppressor p53 in colon cancer cells.

BACKGROUND: In colon cancer, K-Ras oncogenes, which appear to be linked to chemoresistance and poor prognosis, are activated in more than 50% of cases, whereas the tumor suppressor gene p53 is mutationally altered in about 70% of all cases. The transcription factor p53, which is frequently mutated at codon 273, maintains wild-type configuration and possibly carries out residual functions. Although blocking of activated K-Ras may constitute a rational therapeutic concept for this treatment-resistant malignancy, a strategy influencing both oncogenic Ras and the tumor suppressor p53 may be even more promising. MATERIALS AND METHODS: We evaluated the effects of S-trans, trans-farnesyl-thiosalicylic acid (FTS), a novel Ras antagonist on human SW480 and HT-29 colon cancer cells, which both harbor a p53 His273 mutation but express activated K-Ras and wild-type, but overexpressed, H-Ras, respectively. Besides cell growth and morphology, levels of cellular Ras proteins, regulation of p53 and p21(waf1/cip1) expression were analyzed by immunoblotting. The cell cycle arresting potential of FTS was quantified by flow cytometry. RESULTS: We demonstrate that FTS treatment alters the morphology and blocks the growth of SW480 and HT-29 colon cancer cells by both reducing the total amount of Ras and up-regulating the tumor suppressor p53. Furthermore, FTS caused an upregulation of the cyclin-cyclin-dependent kinase (CDK) inhibitor p21(waf1/cip1) and blocked the cell cycle. p53 antisense oligonucleotides not only reduced the level of p53 proteins but correspondingly also blocked the expression of p21(waf1/cip1) in FTS-treated colon cancer cells. CONCLUSIONS: FTS, a unique compound capable of regulating both oncogenic Ras and the tumor suppressor p53 may prove particularly useful for the therapy of colon cancer and other treatment-resistant malignancies where Ras is altered and p53 is either wild-type or mutated in positions that allow residual p53 functions.

Pharmacokinetics and pharmacodynamics of the novel H1-receptor antagonist emedastine in healthy volunteers.

OBJECTIVE: Emedastine is a novel H1-receptor antagonist with pre-clinically well-documented anti-allergic effects. Here, we set out to study the relationship between emedastine pharmacokinetics and the suppressive effect on histamine-induced wheals and flares, and to compare these effects to placebo and cetirizine. METHODS: Emedastine (4 mg q.d.), emedastine (2 mg b.i.d.), cetirizine (10 mg q.d.) and placebo were administered to healthy volunteers in a double-blind, cross-over fashion. On day 1 and day 5 (steady state) following drug administration, wheals and flares were induced by skin-prick testing with 1 mg ml(-1) or 10 mg ml(-1) histamine. RESULTS: Following the administration of 4 mg emedastine q.d., mean area under the concentration-time curve (AUC)0-24 values of 34.49 +/- 24.07 ng h ml(-1) and 47.05 +/- 36.12 ng h ml(-1) were attained on day 1 and day 5, respectively. Following the administration of emedastine (2 mg b.i.d.) mean AUC0-24 values were 29.75 +/- 19.92 ng h ml(-1) and 46.13 +/- 38.50 ng h ml(-1) on day 1 and day 5, respectively. Histamine-induced wheals and flares were significantly more effectively suppressed by emedastine and cetirizine than placebo. At pharmacokinetic steady-state levels, no significant difference could be found in the potency between cetirizine and emedastine (2 mg b.i.d.). CONCLUSION: Emedastine displays pharmacodynamic properties comparable with cetirizine and therefore qualifies as a safe and alternative compound with H1-receptor antagonist properties. Additional larger studies may be needed to substantiate potential benefits of cetirizine over emedastine after single-dose administration.

Deletions of the region 17p11-13 in advanced melanoma revealed by cytogenetic analysis and fluorescence in situ hybridization.

The significance of the p53 tumour-suppressor gene in the oncogenesis of a variety of malignant tumours has been demonstrated over recent years. However, the role of p53 in human malignant melanoma is still unclear. Therefore, we investigated melanoma metastases from 11 patients cytogenetically and with fluorescence in situ hybridization (FISH) after short-term culture, employing a p53 region-specific probe for 17p13.1 and a probe detecting the centromere of chromosome 17. Furthermore, paraffin-embedded tissue samples from nine of these patients were investigated immunohistochemically for expression of the p53 protein. Deletions of the short arm of chromosome 17 were seen in six melanomas in cytogenetic analysis. With FISH, three malignant melanomas had clones with only one p53-allele and an additional four malignant melanomas showed a reduced number of signals at the p53 tumour-suppressor gene locus compared with signals for the centromeric region of chromosome 17. This was confirmed by immunohistochemistry. Our results suggest that the 17p11-13 region is frequently deleted in malignant melanomas and that p53 or other genes located on this band might contribute to the malignant potential of advanced melanoma.

Influence of MUC18/MCAM/CD146 expression on human melanoma growth and metastasis in SCID mice.

The cell surface glycoprotein MUC18MCAM/CD146 was originally defined as a marker of melanoma progression and has been suspected to be directly linked to the metastatic process of this malignancy. In order to address this question, 2 MCAM negative human melanoma cell lines, SK-2 and XP44RO(Mel), were transfected with MCAM-encoding cDNA. Surface MCAM expression on SK-2 and XP44RO(Mel) transfectants was similar to that observed in naturally occurring MCAM positive human melanoma cells and transfectants demonstrated MCAM-dependent increase in homotypic adhesion in vitro. The growth behavior of 7 MCAM transfectants and their respective vector controls was evaluated in SCID mice. Tumor size at 4-5 weeks after s.c. implantation was highly variable, but did not correlate with MCAM expression. Despite massive primary tumor formation at the injection site, no spontaneous metastasis was observed with any of the investigated MCAM transfectants. The influence of MCAM expression on lung metastases formation in an experimental metastasis assay was system dependent, converting only XP44RO(Mel) transfectants into metastatic cells, although increased homotypic adhesion, leading to formation of tumor cell clusters, was observed with transfectants of both cell lines in vitro. Our findings indicate that MCAM expression of human melanoma cells has an influence on later stages of the metastatic process only, namely, extravasation and establishment of new foci of growth, but is per se not sufficient for this process.

Chemosensitisation of malignant melanoma by BCL2 antisense therapy.

BACKGROUND: Chemoresistance of malignant melanoma has been linked to expression of the proto-oncogene BCL2. Antisense oligonucleotides (ASO) targeted against BCL2 mRNA decreased BCL2 protein concentrations, increased tumour-cell apoptosis, and led to tumour responses in a mouse xenotransplantation model when combined with systemic dacarbazine. This phase I-II clinical study investigated the combination of BCL2 ASO (augmerosen, Genasense, G3139) and dacarbazine in patients with advanced malignant melanoma expressing BCL2. METHODS: In a within-patient dose-escalation protocol, 14 patients with advanced malignant melanoma were given augmerosen intravenously or subcutaneously in daily doses of 0.6-6.5 mg/kg plus standard dacarbazine treatment (total doses up to 1000 mg/m2 per cycle). Toxicity was scored by common toxicity criteria. Plasma augmerosen concentrations were assayed by high-performance liquid chromatography. In serial tumour biopsy samples, BCL2 protein concentrations were measured by western blotting and tumour-cell apoptosis was assessed. FINDINGS: The combination regimen was well tolerated, with no dose-limiting toxicity. Haematological abnormalities were mild to moderate. Lymphopenia was common, but no febrile neutropenia occurred. Higher doses of augmerosen were associated with transient fever. Four patients had liver-function abnormalities that resolved within 1 week. Steady-state plasma concentrations of augmerosen were attained within 24 h, and increased with administered dose. By day 5, daily doses of 1.7 mg/kg and higher led to a median 40% decrease in BCL2 protein in melanoma samples compared with baseline, concomitantly with increased tumour-cell apoptosis, which was greatly increased after dacarbazine treatment. Six patients have shown antitumour responses (one complete, two partial, three minor). The estimated median survival of all patients now exceeds 12 months. INTERPRETATION: Systemic administration of augmerosen downregulated the target BCL2 protein in metastatic cancer. Such downregulation of BCL2, combined with standard anticancer therapy, offers a new approach to the treatment of patients with resistant neoplasms.

Novel Ras antagonist blocks human melanoma growth.

During past decades, knowledge of melanoma biology has increased considerably. Numerous therapeutic modalities based on this knowledge are currently under investigation. Advanced melanoma, nevertheless, remains a prime example of poor treatment response that may, in part, be the consequence of activated N-Ras oncoproteins. Besides oncogenic Ras, wild-type Ras gene products also play a key role in receptor tyrosine kinase growth factor signaling, known to be of importance in oncogenesis and tumor progression of a variety of human neoplasms, including malignant melanoma; therefore, it is reasonable to speculate that a pharmacological approach that curtails Ras activity may represent a sensible approach to inhibit melanoma growth. To test this concept, the antitumor activity of S-trans, trans-farnesylthiosalicylic acid (FTS), a recently discovered Ras antagonist that dislodges Ras from its membrane-anchoring sites, was evaluated. The antitumor activity of FTS was assessed both in vitro and in vivo in two independent SCID mouse xenotransplantation models of human melanoma expressing either wild-type Ras (cell line 518A2) or activated Ras (cell line 607B). We show that FTS (5-50 microM) reduces the amounts of activated N-Ras and wild-type Ras isoforms both in human melanoma cells and Rat-1 fibroblasts, interrupts the Ras-dependent extracellular signal-regulated kinase in melanoma cells, inhibits the growth of N-Ras-transformed fibroblasts and human melanoma cells in vitro and reverses their transformed phenotype. FTS also causes a profound and statistically significant inhibition of 518A2 (82%) and 607B (90%) human melanoma growth in SCID mice without evidence of drug-related toxicity. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma and possibly other tumors that either carry activated ras genes or rely on Ras signal transduction more heavily than nonmalignant cells.