Femtosecond X-ray protein nanocrystallography.

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 µm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.

Triple-negative breast cancer possibly transforming into malignant melanoma due to targeted therapy? A case report and review of literature.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by lacking expression of estrogen receptor and progesterone receptor as well as absence of human epidermal growth factor receptor 2 overexpression and is an aggressive clinical phenotype. PATIENTS AND METHODS: We report the case of a 33-year-old woman who has been treated using a targeted approach for TNBC and developed a malignant melanoma metastasis without any primary. RESULTS AND CONCLUSION: Using targeted therapies, tumors can be treated much more effectively, but up to now, we do not know much about potential adverse reactions. Due to the targeted therapy, tumors may be pressurized for transformation. We call for further investigations to rule out the potential risks of targeted therapy in TNBC. This is the first report of a potential transforming of one tumor entity to another by a targeted therapy.

Should grade 3 endometrioid endometrial carcinoma be considered a type 2 cancer-a clinical and pathological evaluation.

OBJECTIVE: Endometrial cancer is classified into: Type I estrogen-dependent endometrioid adenocarcinoma, with good prognosis and type 2 non-estrogen-dependent cancer with serous or clear cell histology and poor prognosis. Grade 3 endometrioid cancers (G3 EEC), share features of type 1 and type 2 cancer and have not been classified as either. This study compares immunohistochemistry and survival in G3 EEC and type 2 cancers. METHODS: Clinicopathological data compared with immunohistochemistry and survival in 156 consecutive patients with poor prognosis cancer-G3 EEC, uterine papillary serous (UPSC) and clear cell carcinoma (CC), sarcoma, carcinosarcoma and endometrial tumors of mixed histology. 131 (84%) datasets were complete, 25 tumors comprising sarcoma, carcinosarcoma or mixed histologies were excluded. Tissue microarray constructed and tested for estrogen receptor (ER), progesterone receptor (PR), p53 and human epidermal growth factor receptor-2 (Her-2). RESULTS: There was no significant difference in the mean age for G3 EEC (n=68) and USPC + CC (n=38), (68.01 and 67.08 respectively, p=0.697) or stage at diagnosis (p=0.384). For ER, PR, p53 and Her-2, there was no significant difference in marker positivity between G3 EEC and UPSC + CC (p=0.612, 0.132, 0.16 and 0.132 respectively). With a mean follow-up time 148 months Disease specific and recurrence-free survival between G3 EEC and USPC + CC was similar (p=0.842 and 0.863). CONCLUSION: G3 EEC and UPSC + CC share similar clinical, immunohistochemistry and poor survival. G3 EEC is better characterised as type 2 cancer and should be treated with similar adjuvant therapy to UPSC/CC.

Viscotoxin and lectin content in foliage and fruit of Viscum album L. on the main host trees of Hyrcanian forests.

Mistletoe (Viscum album L.) is a hemiparasitic plant that absorbs water and nutrients from the host tree. Mistletoe contains two groups of cytotoxic, immunomodulatory and antitumor proteins, viscotoxins and lectins. This study evaluated the quantity and quality of viscotoxins and total lectins in the stems with leaves (foliage) and fruit of mistletoe on Parrotia persica and Carpinus betulus in September with immature green berries and in December with mature white berries. Viscum album L. plants were harvested from host species located in the Hyrcanian forests of Iran in 2019. The highest level of viscotoxins was detected in the December foliage of V. album settled on C. betulus (9.25 mg/g dry weight [DW]), and the highest content of lectins was found in the December foliage of V. album settled on P. persica (0.79 mg/g DW) and C. betulus (0.73 mg/g DW) respectively. The immature green berries of V. album from both host species contained much higher concentrations of viscotoxins and lectins than the mature white berries. Four isoforms of viscotoxins, viscotoxin A1, A2, A3 and B could be identified in all samples of both host species. Viscotoxin A3 was the predominant viscotoxin isoform followed by viscotoxin A1.

Phytochemical analysis and in vitro anti-proliferative activity of Viscum album ethanolic extracts.

BACKGROUND: Viscum album L. (Santalaceae), commonly known as mistletoe, is a hemiparasitic plant traditionally used in complementary cancer treatment. Its antitumor potential is mostly attributed to the presence of aqueous soluble metabolites; however, the use of ethanol as solvent also permits the extraction of pharmacological compounds with antitumor potential. The clinical efficacy of mistletoe therapy inspired the present work, which focuses on ethanolic extracts (V. album "mother tinctures", MT) prepared from different host trees. METHODS: Samples from three European subspecies (album, austriacum, and abietis) were harvested, and five different V. album-MT strains were prepared. The following phytochemical analyses were performed: thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). The proliferation assay was performed with WST-1 after incubation of tumor (Yoshida and Molt-4) and fibroblast cell lines (NIH/3 T3) with different MT concentrations (0.5 to 0.05% v/v). The cell death mechanism was investigated by flow cytometry (FACS) using Annexin V-7AAD. RESULTS: Chemical analyses of MT showed the presence of phenolic acids, flavonoids and lignans. The MT flavonoid and viscotoxin contents (mg/g fresh weight) were highest in Quercus robur (9.67 ± 0.85 mg/g) and Malus domestica (3.95 ± 0.58 mg/mg), respectively. The viscotoxin isoform proportions (% total) were also different among the VA subspecies with a higher content of A3 in V. album growing on Abies alba (60.57 ± 2.13). The phytochemical compounds as well as the viscotoxin contents are probably related to the antitumor effects of MT. The cell death mechanisms evaluated by colorimetric and FACS methodologies involved necrotic damage, which was host tree-, time- and dose- dependent, with different selectivity to tumor cells. Mother tincture from V. album ssp. abietis was the most effective at inducing in vitro cellular effects, even when incubated at the smallest concentration tested, probably because of the higher content of VT A3. CONCLUSION: Our results indicate the promising antitumor potential of Viscum album ethanolic extracts and the importance of botanical and phytochemical characterization for in vitro anti-proliferative effects.

Circulating vascular endothelial growth factors and their soluble receptors in pre-invasive, invasive and recurrent cervical cancer.

AIM: To study the impact of circulating vascular endothelial growth factors (VEGF) -A, -C and -D and their soluble receptors VEGFR-1/-2 on disease invasion and progression in patients with pre-invasive (CIN), invasive (PCC) and recurrent (RCC) cervical cancer. PATIENTS AND METHODS: Blood samples were obtained from 125 women, including 50 cases of CIN, 51 of PCC and 24 of RCC, before treatment. Soluble (s) biomarker levels were determined by ELISA and tested for correlation with histopathological factors. RESULTS: With disease progression, sVEGF-A (p = 0.007) and sVEGFR-2 (p = 0.014) significantly increased, while sVEGF-D (p = 0.046) decreased. sVEGFR-2 levels were increased in node+ patients (p = 0.024) and in metastatic disease (p = 0.003). sVEGF-A values were higher in HPV+ cases (p = 0.019). In detecting disease invasiveness, sensitivity and specificity were 76% and 48% for sVEGF-A, 52% and 32% for sVEGF-D, 25% and 94% for sVEGF-C, 93% and 6% for sVEGFR-1 and 73% and 34% for sVEGFR-2, respectively. CONCLUSION: In cervical neoplasia, a switch from a lymphangiogenic phenotype towards a hemangiogenic phenotype occurs with disease invasion and progression. The sensitivity and specificity values, however, seem not convincing enough to establish these factors as clinical markers for disease invasiveness in cervical cancer.

Transfection of keratin 18 gene in human breast cancer cells causes induction of adhesion proteins and dramatic regression of malignancy in vitro and in vivo.

This study shows that high keratin 18 (K18) expression in tumor cells is associated with reduced invasiveness in vitro and lack of tumorigenicity in nude mice. We previously showed that high K18 expression correlated with a good prognosis and that reducing K18 expression increased the aggressiveness of established breast cancer cell lines. To confirm these observations, we transfected the human K18 gene into the human breast cancer cell line MDA-MB-231 and isolated a stable overexpressing clone. The forced K18 expression was associated with a complete loss of the previously strong vimentin expression in the parent cell line, induction of the K18 dimerization partner K8, and up-regulation of adhesion proteins. These changes were accompanied by a dramatic reduction in the aggressiveness of the K18 transfectants in vitro and in vivo. We conclude that forced reexpression of K18 causes at least partial redifferentiation of the tumor cell, followed by a corresponding regression of malignant phenotype.

Epidermal growth factor receptor changes during breast cancer metastasis.

BACKGROUND: Since the anti-HER2 monoclonal antibody trastuzumab made its triumphant advance into breast cancer therapy, new selective agents, including pan-HER inhibitors are entering clinical practice. MATERIALS AND METHODS: This study investigates the expression of the four HER-family members, HER1-4, in 48 primary breast carcinomas (PBC) and corresponding distant metastases (CDM) by immunohistochemistry and fluorescence in situ hybridisation. RESULTS: Concordance rate between PBC and CDM was 79% for HERI and HER2, 67% for HER3 and 56% or HER4. Expression of HER1-3 was associated with poor prognosis compared to HER-negative disease (p = 0.036). HER4 overexpression was associated with a better outcome (p = 0.003). CONCLUSION: Though most tumours demonstrate a stable HER expression pattern, both loss and acquisition of HER receptor overexpression can occur during metastasis. HER4 overexpression predicts prolonged survival compared to receptor negative disease, while the opposite is true for HER1-3. Consequences for modem antibody therapy are discussed.

The prognostic significance of epithelial-mesenchymal transition in breast cancer.

BACKGROUND: This study investigates the influence of epithelial-mesenchymal transition on prognosis in breast cancer. MATERIALS AND METHODS: Eighty breast carcinomas as well as 6 breast cancer cell lines were analysed immunohistochemically for the expression of epithelial keratins (K) K8, K19 and mesenchymal vimentin. Protein expression was correlated with histopathological factors and clinical follow-up. RESULTS: Suppression of K8 and K19 occurred in the majority of the tumours (72.5% and 65%), while aberrant expression of vimentin was found in 21.2% of the tumours. Suppression of K8 as well as expression of vimentin was significantly correlated with short survival (p < 0.004 and p < 0.006). Moreover HER2 overexpression significantly correlated with K19 (p < 0.0004) and vimentin (p < 0.0005). In cell lines with increasing invasive potential, epithelial keratins were lost in favour of mesenchymal vimentin. CONCLUSION: The transition from epithelial keratin to mesenchymal vimentin expression marks an important step in the malignant progression of breast cancer.

Phase II study of capecitabine plus trastuzumab in human epidermal growth factor receptor 2 overexpressing metastatic breast cancer pretreated with anthracyclines or taxanes.

PURPOSE: The oral fluoropyrimidine carbamate, capecitabine, is a highly active and well-tolerated treatment for metastatic breast cancer. In patients treated previously with anthracyclines and taxanes, capecitabine is an approved single-agent therapy. Trastuzumab, a monoclonal antibody targeting the human epidermal growth factor receptor 2 (HER-2), is also highly active in HER-2-overexpressing breast cancer. We have conducted a phase II study to confirm activity and feasibility of capecitabine and trastuzumab in combination in HER-2-overexpressing advanced/metastatic breast cancer. PATIENTS AND METHODS: Twenty-seven patients with HER-2-overexpressing metastatic breast cancer previously treated with anthracyclines and/or taxanes received oral capecitabine 1,250 mg/m(2) bid for 14 days followed by a 7-day rest period combined with intravenous trastuzumab 4 mg/kg body weight on day 1 (loading dose) followed by 2 mg/kg weekly. RESULTS: Capecitabine/trastuzumab treatment achieved objective responses in 12 patients (45%), including complete response in four patients (15%) and partial response in eight patients (30%). Disease was stabilized in an additional nine patients (33%). The median overall survival time was 28 months, and the median progression-free survival time was 6.7 months. The safety profile of the combination was favorable and predictable, with a low incidence of grade 3/4 adverse events. The most common adverse events were pain, hand-foot syndrome, and GI toxicities. Severe myelosuppression was rare and severe alopecia did not occur. CONCLUSION: These data confirm that the combination of capecitabine and trastuzumab is highly active in patients with HER-2-overexpressing anthracycline- and/or taxane-pretreated breast cancer, with only slight restrictions regarding quality of life.

Viscotoxins, mistletoe lectins and their isoforms in mistletoe (Viscum album L.) extracts Iscador.

The purpose of the following study was to evaluate the presence of the most frequently investigated pharmacologically active mistletoe compounds, viscotoxins (VT) and mistletoe lectins (ML), in European mistletoe Viscum album L. and in the pharmaceutical mistletoe preparations Iscador. Quantitative analysis of the VT isoforms A1, A2, A3, B, 1-PS, and U-PS in fresh mistletoe plant material from the three European subspecies of V. album, during fermentative extraction of mistletoe and in Iscador showed that the pharmaceutical proceeding specific for the preparation of Iscador warrants a high yield of VT. No degradation or transformation of VT during the production process became apparent. The VT compositions of the three host specific European subspecies of V. album, ssp. album, ssp. abietis, and ssp. austriacum, showed characteristic differences. They ensured the identification of the subspecies specific types of Iscador. ML contents of mistletoe extracts were reduced during fermentative extraction. The quantified contents of total ML were 261 +/- 9.3 ng/ml in Iscador M 5 mg spec. and 391 +/- 18.3 ng/ml in Iscador Qu 5 mg spec. Binding of ML to the glycoprotein asialofetuin (type 1) was found to be temperature dependent. Binding activity was increased to 250 % (ML I) and 410 % (ML II and ML III) respectively by decreasing temperature from 30 degrees C to 4 degrees C. 95% of ML could be eliminated from Iscador by affinity chromatography with immobilised glycoproteins at 0 degrees C. Quantitative extraction of ML from the crude extract and their analysis by SDS-PAGE revealed the presence of about 30 % ML I, 20% ML II, and 50% ML III in Iscador M 5 mg spec. and Iscador Qu 5 mg spec. The annual course of concentrations of ML and VT in the leaves of V. album showed maximal ML contents in December and culminaton of VT in June. Seasonal fluctuations of the composition of mistletoe imply the importance of fixed harvesting seasons.

Analysis of HER2 and HER4 in human myocardium to clarify the cardiotoxicity of trastuzumab (Herceptin).

PURPOSE: When combined with anthracyclines, the humanized anti-HER2 monoclonal antibody trastuzumab (Herceptin) provides significant clinical benefit for women with HER2-overexpressing metastatic breast cancer. However, its use is limited by severe cardiotoxicity. To clarify whether myocardial HER2 and HER4 expression in response to anthracycline exposure and cardiac damage contributes to cardiotoxicity, we assessed expression of HER2 and HER4 in pathologically altered myocardium. EXPERIMENTAL DESIGN: Cardiac biopsies from 60 patients with severe heart disease and cardiac tissue from 35 patients with breast cancer were obtained. Twenty-five of the patients with breast cancer had previously received anthracyclines. Three of 10 anthracycline-naïve patients with breast cancer had received trastuzumab. Expression of HER2 and HER4 was analyzed immunohistochemically (HER2: HercepTest/A0485 (Dako), Cy3 detection (Dianova); HER4: Ab-4 (NeoMarkers)). FISH analysis (Ventana) was used to assess HER2 gene amplification. RESULTS: Immunohistochemistry revealed weak HER2 membrane staining in six cardiac biopsies, appearing as dotted staining of the whole cell membrane and intensified HER2 signal using fluorescent Cy3 labeling. No HER2 membrane staining was detected in the remaining 54 cardiac biopsies or in the myocardium of the 35 patients with breast cancer. HER2 gene amplification was not observed. All specimens showed the mild cytoplasmatic HER4 staining of normal myocardium. No strong HER4 expression was detected. CONCLUSIONS: Cardiac alterations are not associated with an strong increase in HER2 and HER4 levels. IHC detects potential low-level HER2 expression in some samples. However, a more sensitive technique may be needed for studies of the role of HER2 in cardiac tissue. These data do not exclude a role for inhibition of cardiac HER2 expression by trastuzumab in the onset of heart failure in trastuzumab-treated patients.

Comparative membrane interaction study of viscotoxins A3, A2 and B from mistletoe (Viscum album) and connections with their structures.

Viscotoxins A2 (VA2) and B (VB) are, together with viscotoxin A3 (VA3), among the most abundant viscotoxin isoforms that occur in mistletoe-derived medicines used in anti-cancer therapy. Although these isoforms have a high degree of amino-acid-sequence similarity, they are strikingly different from each other in their in vitro cytotoxic potency towards tumour cells. First, as VA3 is the only viscotoxin whose three-dimensional (3D) structure has been solved to date, we report the NMR determination of the 3D structures of VA2 and VB. Secondly, to account for the in vitro cytotoxicity discrepancy, we carried out a comparative study of the interaction of the three viscotoxins with model membranes. Although the overall 3D structure is highly conserved among the three isoforms, some discrete structural features and associated surface properties readily account for the different affinity and perturbation of model membranes. VA3 and VA2 interact in a similar way, but the weaker hydrophobic character of VA2 is thought to be mainly responsible for the apparent different affinity towards membranes. VB is much less active than the other two viscotoxins and does not insert into model membranes. This could be related to the occurrence of a single residue (Arg25) protruding outside the hydrophobic plane formed by the two amphipathic alpha-helices, through which viscotoxins are supposed to interact with plasma membranes.

Differences in T-cell immunity toward tumor-associated antigens in colorectal cancer and breast cancer patients.

There is increasing evidence that tumors elicit specific T-cell responses in a substantial proportion of patients. Recently, we have shown that in patients with colorectal cancer specific T cells against the tumor-associated antigens (TAA) Ep-CAM, her-2/neu or CEA can be detected in peripheral blood using IFNgamma-ELISPOT assay. In our study, we have analyzed T-cell responses against HLA-A*0201-restricted epitopes of these TAA in peripheral blood of patients with breast cancer and colorectal cancer. Surprisingly, a complete absence of ex vivo T-cell responses against these TAA was found in 20 patients with breast cancer. In contrast, specific T cells were detectable in 12 of 49 patients with colorectal cancer against at least 1 of these TAA, confirming our previous results. T-cell responses against influenza-derived peptides were similar in both malignancies. The results of our study indicate a difference either of tumor immunogenicity or of the migratory pattern of tumor-specific T cells between breast cancer and colorectal cancer patients. The findings reported here have implications for the development of antigen-specific T-cell therapies.

NMR solution structure of viscotoxin C1 from Viscum album species Coloratum ohwi: toward a structure-function analysis of viscotoxins.

The high resolution three-dimensional structure of the newly discovered plant viscotoxin C1, from the Asiatic Viscum album ssp. Coloratum ohwi, has been determined in solution by (1)H NMR spectroscopy at pH 3.6 and 285 K. The viscotoxin C1-fold, consisting of a helix-turn-helix motif and a short stretch of an antiparralel beta-sheet is very similar to that found for the highly similar viscotoxins A2 and A3 and for other related thionins. Different functional properties of members of the thionin family are discussed here in light of the structural and electrostatic properties. Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from the other members because of its high toxicity against tumoral cells. Key residues for the modulation of viscotoxin cytotoxicity have been identified on the basis of sequence and structural alignment.

Oxaliplatin and 5-fluorouracil for heavily pretreated metastatic breast cancer: a preliminary phase II study.

UNLABELLED: Oxaliplatin shows in vitro and in vivo synergism with 5-fluorouracil (5-FU). In this study we evaluate the clinical efficacy of oxaliplatin and 5-FU in heavily pretreated metastatic breast cancer. Eligible patients had to be pretreated with both anthracyclines and taxanes. Pretreatment with capecitabine was recommended but not mandatory. Chemotherapy: oxaliplatin 85 mg/m2/2 h day 1, folinic acid 400 mg/m2/2 h day 1, 5-FU 400 mg/m2 i.v. push day 1, 5-FU 2400 mg/m2 continuous infusion/48 h day 1, q2w. Fourteen patients were included: one male and 13 females; age: median 53 years (38-62); ECOG 0: three patients, 1: nine patients, 2: two patients; all patients were pretreated with anthracyclines and taxanes, capecitabine: nine patients, vinorelbine: six patients, trastuzumab: four patients, hormonal therapy: 12 patients; lines of prior palliative chemotherapy: 0: one patient, 1: three patients, 2: one patient, 3: three patients, 4: four patients, 5: two patients. RESULTS: median number of cycles: 8 (range 1-17). Toxicity (14 patients evaluable; no. of patients with Common Toxicity Criteria grade 3/4): asthenia: 2/-, paraesthesia 3/-, leuko/neutropenia: 1/2, no neutropenic fever, other (alopecia, skin): 2/-. Response (12 patients evaluable, all with bidimensionally measurable disease): complete remission: no patients, partial remission: four patients (33%, all confirmed), stable disease: five patients, progressive disease: three patients. We conclude that despite the small number of patients, the combination of 5-FU and oxaliplatin shows promising efficacy in this heavily pretreated population.