Dariusz Nejc

Parthenolide, a sesquiterpene lactone from the medical herb feverfew, shows anticancer activity against human melanoma cells in vitro.

Metastatic melanoma is a highly life-threatening disease. The lack of response to radiotherapy and chemotherapy highlights the critical need for novel treatments. Parthenolide, an active component of feverfew (Tanacetum parthenium), inhibits proliferation and kills various cancer cells mainly by inducing apoptosis. The aim of the study was to examine anticancer effects of parthenolide in melanoma cells in vitro. The cytotoxicity of parthenolide was tested in melanoma cell lines and melanocytes, as well as melanoma cells directly derived from a surgical excision. Adherent cell proliferation was measured by tetrazolium derivative reduction assay. Loss of the plasma membrane integrity, hypodiploid events, reactive oxygen species generation, mitochondrial membrane potential dissipation, and caspase-3 activity were assessed by flow cytometric analysis. Microscopy was used to observe morphological changes and cell detachment. Parthenolide reduced the number of viable adherent cells in melanoma cultures. Half maximal inhibitory concentration values around 4 μmol/l were determined. Cell death accompanied by mitochondrial membrane depolarization and caspase-3 activation was observed as the result of parthenolide application. Interestingly, the melanoma cells from vertical growth phase and melanocytes were less susceptible to parthenolide-induced cell death than metastatic cells when drug concentration was at least 6 μmol/l. Reactive oxygen species level was not significantly increased in melanoma cells. However, preincubation of parthenolide with the thiol nucleophile N-acetyl-cysteine protected melanoma cells from parthenolide-induced cell death suggesting the reaction with intracellular thiols as the mechanism responsible for parthenolide activity. In conclusion, the observed anticancer activity makes parthenolide an attractive drug candidate for further testing in melanoma therapy.

Long-term survival of endometrioid endometrial cancer patients

Introduction To establish risk factors for onset and progression of endometrioid endometrial cancer still remains the aim of scientists. The aim of the study was to determine disease-free survival (DFS) and overall survival (OS) in women with endometrioid endometrial cancer. Material and methods A retrospective review of 142 patients with endometrioid endometrial cancer after surgery treated with adjuvant radiotherapy and/or chemotherapy in the Regional Cancer Centre in Lodz between 2002 and 2004 was performed. Clinical and pathological data were correlated with clinical outcome and survival. Results In 3 patients (2.1%) clinical progression was diagnosed during the treatment. In 23 patients (16.7%) after primary remission, relapse was diagnosed 2-56 months after treatment. DFS and OS were 81.7% and 83.1% respectively. Better DFS significantly correlated with larger number of pregnancies (> 1), stage I of the disease and optimal surgery. Lower stage of disease, pelvic lymph node dissection, optimal surgery and depth of myometrial infiltration ≤ 50% were independent prognostic factors for better OS. Conclusions The results of our study provided significant evidence that early detection of endometrioid endometrial cancer enables optimal surgery. It reduces the indications for adjuvant therapy in stage I of the disease, and makes the prognosis significantly better. Other clinical and pathological factors such as numerous pregnancies, pelvic lymphadenectomy, and depth of myometrial infiltration, although important, are of less significance. Further prospective, randomized studies are necessary to prove the role of these factors.

The first description of sentinel node biopsy in a patient with amelanotic melanoma of the glans penis

We present the first description of sentinel node biopsy in a patient with amelanotic melanoma of the glans penis. The patient underwent partial amputation of the penis due to tumor of the glans. Pathologic examination of the postoperative specimen revealed the presence of a very rare malignancy--amelanotic melanoma. Sentinel node biopsy, with the use of the combined radiotracer/blue dye technique, was performed. Preoperative lymphoscintigraphy was performed the day before surgery. During surgery, blue dye mapping and intraoperative detection of gamma radiation were used. Two sentinel nodes were identified in the left inguinal region and one sentinel node in the right inguinal region. All sentinel nodes were an intense violet color; in each case, the level of radiation in the sentinel node was almost 20 times higher than the level of radiation in the node bed. Routine hematoxylin and eosin staining and immunohistochemistry (HMB-45) revealed the presence of micrometastasis in one of the sentinel nodes harvested from the left inguinal region. Consequently, left inguinal, iliac and obturatory lymphadenectomies were performed. The final pathologic examination revealed the presence of one metastasis (diameter, 2 mm) in one of the resected non-sentinel nodes. No relapse has been observed during 18 months of follow-up.

Parthenolide induces MITF-M downregulation and senescence in patient-derived MITF-M high melanoma cell populations

The activity of the M isoform of microphthalmia-associated transcription factor (MITF-M) has been attributed to regulation of differentiation, proliferation, survival and senescence of melanoma cells. MITF expression was shown to be antagonized by the activation of transcription factor NF-κB. Parthenolide, an inhibitor of NF-κB, has not been yet reported to affect MITF-M expression. Our results obtained in patient-derived melanoma cell populations indicate that parthenolide efficiently decreases the MITF-M level. This is neither dependent on p65/NF-κB signaling nor RAF/MEK/ERK pathway activity as inhibition of MEK by GSK1120212 (trametinib) and induction of ERK1/2 activity by parthenolide itself do not interfere with parthenolide-triggered depletion of MITF-M in both wild-type BRAF and BRAFV600E melanoma populations. Parthenolide activity is not prevented by inhibitors of caspases, proteasomal and lysosomal pathways. As parthenolide reduces MITF-M transcript level and HDAC1 protein level, parthenolide-activated depletion of MITF-M protein may be considered as a result of transcriptional regulation, however, the influence of parthenolide on other elements of a dynamic control over MITF-M cannot be ruled out. Parthenolide induces diverse effects in melanoma cells, from death to senescence. The mode of the response to parthenolide is bound to the molecular characteristics of melanoma cells, particularly to the basal MITF-M expression level but other cell-autonomous differences such as NF-κB activity and MCL-1 level might also contribute. Our data suggest that parthenolide can be developed as a drug used in combination therapy against melanoma when simultaneous inhibition of MITF-M, NF-κB and HDAC1 is needed.

Significance of Bax expression in breast cancer patients.

UNLABELLED Bax protein, the proapoptotic member of Bcl-2 protein family, plays the key role in apoptosis pathway. THE AIM OF THE STUDY was to assess the expression of Bax protein in breast cancer cells. MATERIAL AND METHODS Sixty-two breast cancer patients were included in the study. The control group encompassed 11 fibroadenoma patients. Single cells were isolated from defrosted samples and prepared for flow cytometry measurement. RESULTS Median expression of Bax protein in study group was 7.9% (range: 0-49.4%) and was significantly lower than in control (median expression 15.8%; range 4.9-30.9%; p=0.034). Expression of Bax correlated with expression of p53 and caspase-3 proteins (p<0.01, rank Spearman test). In patients under 70 years old and with positive estrogen receptors status the expression of Bax protein was significantly higher (p=0.03 and p=0.01 respectively). CONCLUSIONS Lower expression of Bax protein in breast cancer cells may suggest the potential way of apoptosis avoidance of tumor cells. Correlations among Bax protein, p53 and caspase-3 are likely associated with active apoptotic mechanism in breast cancer cells expressing Bax protein. Further investigation with long time follow-up should be performed to establish the prognostic role of Bax protein expression in breast cancer patients.

Recurrence of cholangiogenous carcinoma in port-sites two years after laparoscopic removal of noncancerous gallbladder

We present a unique case of carcinoma diagnosed in port-site, two years after uncomplicated laparoscopic cholecystectomy for benign cholecystitis. Analysis of morphology and cytokeratin profile (CK19+ and CK20+/-) of resected port-site tumor allows us to establish the diagnosis of tubular carcinoma with probable cholangiogenic origin. The primary carcinoma was not diagnosed in archival gallbladder tissue, despite repeated histological examination. No other primary tumor was identified during follow-up. Patient history and histological/immunohistochemical picture of the recurrent tumor suggested that primary carcinoma was probably located in the gallbladder, but was not detected during initial and repeated histological examinations of postoperative specimen. The patient is still alive, 12 months after the first port-site recurrence and 36 months after initial laparoscopy.

Exogenous growth factors bFGF, EGF and HGF do not influence viability and phenotype of V600EBRAF melanoma cells and their response to vemurafenib and trametinib in vitro

It has been shown that the response of V600EBRAF melanoma cells to targeted therapeutics is affected by growth factors. We have investigated the influence of three different growth factors, bFGF, EGF and HGF used either alone or in combination, on the response of V600EBRAF melanoma cell populations established from surgical specimens to vemurafenib and trametinib, targeting V600EBRAF and MEK1/2, respectively. We report that proliferation and phenotype of V600EBRAF melanoma cell populations were not detectably influenced by exogenous growth factors. Neither cell distribution in cell cycle and CCND1 expression nor activity of signaling pathways crucial for melanoma development and maintenance, including the RAF/MEK/ERK pathway, WNT/β-catenin pathway and NF-κB signaling, were affected by the presence of different growth factors. We furthermore show that vemurafenib and trametinib abrogated the activity of ERK1/2, arrested cells in G0/G1 cell cycle phase, triggered apoptosis, induced changes in the expression of CXCL8, CCND1 and CTGF and the frequency of Ki-67high and CD271high cells. These effects were, however, similar in the presence of different growth factors. Interestingly, comparable results were also obtained for melanoma cells grown without exogenous growth factors bFGF, EGF and HGF for a period as long as 4 months prior the drug treatment. We conclude that the composition or lack of exogenous growth factors bFGF, EGF and HGF do not markedly influence viability and phenotype of V600EBRAF melanoma cells and their response to vemurafenib and trametinib in vitro. Our results question the necessity of these growth factors in the medium that is used for culturing V600EBRAF melanoma cells.

Ex vivo Search for Sentinel Node in Postmastectomy Specimens: Should We Use a Transverse Incision for Mastectomy?

BackgroundAccording to the concept of sentinel node (SN), the lymphatic pathway leading to SN should be regarded as the main and the most important lymphatic route from primary tumor to regional lymph nodes. We performed ex vivo blue-dye SN mapping in postmastectomy specimens to assess whether the main lymphatic tract leading to SN is completely removed during mastectomy. We assumed that ex vivo identification of SN may be possible only if the entire lymphatic tract leading to sentinel node is removed from within the postmastectomy specimen.MethodsBlue dye (1 mL) was injected intracutaenously, periareolary into each of 28 postmastectomy specimens. In 13 cases mastectomy was performed with the use of transverse skin incision; in 15 cases oblique incision was used.ResultsThe use of transverse skin incision during modified radical mastectomy allowed identification of the sentinel node and removal of the entire lymphatic pathway leading to sentinel node only in 4 of 15 cases (31%). Conversely, the use of oblique skin incision during modified radical mastectomy allowed identification of the sentinel node and removal of the entire lymphatic pathway leading to sentinel node in 12 of 15 cases (80%).ConclusionsOur experiment revealed that the use of transverse skin incision during modified radical mastectomy may not be the best choice for breast cancer patients. In our opinion, this observation may be especially important for patients not irradiated postoperatively.