Leszek Kozlowski

Lactacystin Inhibits Cathepsin A Activity in Melanoma Cell Lines

We describe the inhibitory effect of the proteasome inhibitor, lactacystin, on cathepsin A activity in murine melanoma cell lines. In vitro lactacystin metabolite, β-lactone, at a concentration of 1 µM, significantly suppressed cathepsin A activity in B78 melanoma cell lysates by about 50%. Exposure of three murine melanoma cell lines with different metastatic potential to lactacystin at a concentration of 5 µM for 6 h caused a significant reduction in the carboxypeptidase activity of this enzyme, while the inhibitory activity remained unchanged for at least 12 h. Other proteasome-specific inhibitors, e.g. epoxomicin and N-benzyloxycarbonyl-Ile-Glu(O-tert-Bu)-Ala-leucinal (PSI) at a concentration of 1 µM did not affect cathepsin A activity in melanoma cell line lysates. These data support our previous proposal that lactacystin is not a specific inhibitor of the proteasome. Since cathepsin A is also a tumor-associated enzyme, further research is needed to clarify its role and the significance of its inhibition by lactacystin in tumor biology.

The significance of the expression of ERRα as a potential biomarker in breast cancer

It was shown the functional crosstalk between ERRalpha and ERalpha in breast cancer, however, the biological significance of estrogen-related receptor alpha (ERRalpha) remains largely unclear. Therefore, we examined the expression of ERRalpha in 39 primary human breast cancer tissues and 19 matched normal tissues using RT-PCR and immunohistochemistry in the context of the aromatase, ERalpha and proliferation markers (c-myc, Ki-67) expression. Compared to the normal breast tissue, breast cancer tissues showed a slightly higher expression level of ERRalpha mRNA (mean 46.2+/-S.D.42.0, 57.7+/-S.D.58.7, respectively). However, ERRalpha mRNA levels in breast cancer tissues showed greater diversity than in normal tissues. Immunohistochemical analysis of breast cancers revealed perinuclear and cytoplasmic localization of ERRalpha. Our study shows that there is no correlation between ERRalpha and ERalpha expression. We demonstrated a positive correlation between ERRalpha and c-myc at the transcriptional level and statistically significant positive correlation between aromatase and the ERRalpha at protein level. It seems that ERRalpha could play an important role in the alternative pathway to classical estrogen receptors-dependent pathway in cell signaling. Development and use of ERRs modulators might lead in the future to design new well-tolerated and individualized therapeutic agents.

Immunohistochemical study of KiSS1 and KiSS1R expression in human primary breast cancer: Association with breast cancer receptor status, proliferation markers and clinicopathological features.

Recent studies have raised doubts about the protective role of KiSS1/KiSS1R in breast malignancy progression. However, the role of the KiSS1/KiSS1R system in primary breast cancer remains largely unknown. The aim of the present study was to characterize the biology and invasiveness potential of primary breast cancer through evaluation of KiSS1/KiSS1R protein expression and cellular localization with regard to lymph node metastasis status, receptor status (ERs, PR and HER-2/neu), and expression of aromatase, MMP-9, Ki-67 and Cyclin D1 in primary invasive breast cancer tissues. We showed increased protein expression of both KiSS1/KiSS1R and MMP-9 in the cancerous tissues compared with noncancerous tissue adjacent to the breast tumour. In the studied group of breast cancer samples, we observed a positive correlation between KiSS1 and MMP-9. We also showed a positive correlation between KiSS1R and aromatase expression in all studied breast cancers. We did not notice any associations between system and cell cycle regulators. KiSS1/KiSS1R did not correlate either with Cyclin D1 and Ki-67 or with receptor status. However, we showed higher levels of KiSS1R expression in ERα-negative cases than in ERα-positive cases in patients with lymph node metastasis. Present data do not confirm the protective role of KiSS1/KiSS1R in breast cancer progression, but our results do support the hypothesis that the KiSS1/KiSS1R system is activated even in primary breast cancer and sustained during invasion to local lymph nodes.

Loss of heterozygosity (LOH)--implications for human genetic identification.

The aim of this study was assessment of possible effects of loss of heterozygosity on human genetic identification of histolopathogical tissue sections. DNA templates were extracted from tumour tissue specimens excised from oncological patients and from reference blood samples. AmpFlSTR Identifiler PCR Amplification Kit and ABI 310 Genetic Analyzer (Applera) were used to obtain genetic profiles. Frequency of LOH was calculated for respective samples. Fishers exact test was performed for statistical analysis. Forty-two percent of the 101 cancer cases analysed were found to possess alterations of the microsatellites manifesting with allelic loss. The most frequently altered loci were D3S1358 and D18S51. The alteration was detected in 47% of cases with larynx carcinoma, 44% of cases with uveal melanoma, 60% of cases with cervical cancers, one case of liposarcoma G3 and one case od neurofibrosarcoma. No LOH was found in liposarcoma G1, dermatofibrosarcoma and cystosarcoma protuberans in either primary or recurrent tumours. In benign tumours (lipoma and fibroma) LOH was also absent. During genotyping of DNA extracted from histopathological tissue sections caution should be taken when non-match or exclusion based on few discrepancies is concluded.

Loss of heterozygosity on chromosomes 3p, 18q21.3 and 11p15.5 as a poor prognostic factor in stage II and III (FIGO) cervical cancer treated by radiotherapy

Loss of heterozygosity (LOH) has been shown to be an important prognostic factor in a variety of malignant neoplasms. Cervical cancer develops as result of multiple genetic alterations. The aim of this study was to analyze presence of LOH in cervical cancer and to identify the correlation between LOH and survival and relapse-free survival time in patients treated with radiotherapy. Studies were performed on tumor specimens and venous blood from 20 patients with cervical cancer (squamous cell carcinoma G2 and G3) in stage II and III (FIGO) treated with radiotherapy. DNA was isolated using organic extraction. Additional microcolumn purification was performed. The fluorescent multiplex polymerase chain reaction (PCR) was used to amplify 10 microsatellite loci included in commercially available human identification kits. Microsatellite marker BAT 26 was amplified in separate PCR reactions. 75% cervical cancers manifested LOH. LOH in BAT 26 analysis (chromosome 2) was present in all these specimens. 60% of the cases showed LOH at one or more of other examined loci (mostly on 3p, 18q21.3, and 11p15.5). Eight of nine cervical cancers in clinical stage III showed LOH. All cases of G3 squamous cell carcinoma of the cervix manifested LOH on 2p. Patients with LOH have worse prognosis for survival and relapse-free survival compared to patients without LOH.