Melanocortin receptor 4 as a new target in melanoma therapy: Anticancer activity of the inhibitor ML00253764 alone and in association with B-raf inhibitor vemurafenib.

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor. The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed. Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity. Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies.

A Luminex Approach to Develop an Anti-Tumor-Associated Antigen Autoantibody Panel for the Detection of Prostate Cancer in Racially/Ethnically Diverse Populations.

(1) Background: Autoantibodies to tumor-associated antigens (TAAs) have emerged as promising cancer biomarkers. Luminex technology offers a powerful approach for the simultaneous detection of multiple anti-TAA autoantibodies. (2) Methods: We aimed to utilize Luminex technology to evaluate and optimize a panel of anti-TAAs autoantibodies for detecting prostate cancer (PCa), which included autoantibodies to fourteen TAAs. A total of 163 serum samples (91 PCa, 72 normal controls) were screened to determine the levels of the autoantibodies using the Luminex assay. (3) Results: Twelve autoantibodies exhibited significantly high frequencies ranging from 19.8% to 51.6% in the PCa group. Receiver operating characteristic (ROC) curve analysis revealed area under the curve (AUC) values ranging from 0.609 to 0.868 for the twelve autoantibodies individually. We further confirmed the performance of the HSP60 autoantibody by using an enzyme-linked immunosorbent assay (ELISA) in a larger sample comprising 200 PCa sera, 20 benign prostatic hyperplasia (BPH) sera, and 137 normal control sera. The results obtained from the Luminex assay were consistent with the ELISA findings. We developed a panel consisting of three autoantibodies (p16, IMP2, and HSP60) which achieved an impressive AUC of 0.910 with a sensitivity of 71.4% and a specificity of 95.8%. The panel was also evaluated in PCa patients from different races/ethnicities with the best performance observed in distinguishing the Hispanic American patients with PCa from normal controls. (4) Conclusions: We developed an anti-TAA autoantibody panel for the detection of PCa that exhibits promising performance. This panel holds significant potential as a high-throughput tool to facilitate PCa detection.

ErbB2/Her2-dependent downregulation of a cell death-promoting protein BLNK in breast cancer cells is required for 3D breast tumor growth.

A significant proportion of breast cancers are driven by ErbB2/Her2 oncoprotein that they overexpress. These malignancies are typically treated with various ErbB2-targeted drugs, but many such cancers develop resistance to these agents and become incurable. Conceivably, treatment of ErbB2-positive cancers could be facilitated by use of agents blocking oncogenic signaling mechanisms downstream of ErbB2. However, current understanding of these mechanisms is limited. The ability of solid tumor cells to resist anoikis, cell death triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. In an effort to understand the mechanisms of ErbB2-driven breast cancer cell anoikis resistance we found that detachment of non-malignant breast epithelial cells from the ECM upregulates a cell death-promoting tumor suppressor adapter protein BLNK and that ErbB2 blocks this upregulation by reducing tumor cell levels of transcription factor IRF6. We further observed that trastuzumab, a therapeutic anti-ErbB2 antibody, upregulates BLNK in human trastuzumab-sensitive but not trastuzumab-resistant ErbB2-positive breast cancer cells. Moreover, we established that BLNK promotes anoikis by activating p38 MAP kinase and that ErbB2-dependent BLNK downregulation blocks breast cancer cell anoikis. In search for pharmacological approaches allowing to upregulate BLNK in tumor cells we found that clinically approved proteasome inhibitor bortezomib upregulates IRF6 and BLNK in human breast cancer cells and inhibits their 3D growth in a BLNK-dependent manner. In addition, we found that BLNK upregulation in human ErbB2-positive breast cancer cells blocks their ability to form tumors in mice. Furthermore, we used publicly available data on mRNA levels in multiple breast cancers to demonstrate that increased BLNK mRNA levels correlate with increased relapse-free survival in a cohort of approximately 400 patients with ErbB2-positive breast cancer. In summary, we discovered a novel mechanism of ErbB2-driven 3D breast tumor growth mediated by ErbB2-dependent BLNK downregulation.

Cyclic Metronomic Chemotherapy for Pediatric Tumors: Six Case Reports and a Review of the Literature.

We report a retrospective case series of six Hispanic children with tumors treated with metronomic chemotherapy. The six cases comprised one rhabdoid tumor of the kidney, one ependymoma, two medulloblastomas, one neuroblastoma, and a type II neurocytoma of the spine. Treatment included oral cyclophosphamide daily for 21 days alternating with oral etoposide daily for 21 days in a backbone of daily valproic acid and celecoxib. In one case, celecoxib was substituted with sulindac. Of the six patients, three showed complete responses, and all patients showed some response to metronomic therapy with only minor hematologic toxicity. One patient had hemorrhagic gastritis likely associated with NSAIDs while off prophylactic antacids. These data add to a growing body of evidence suggesting that continuous doses of valproic acid and celecoxib coupled with alternating metronomic chemotherapy of agents such as etoposide and cyclophosphamide can produce responses in pediatric tumors relapsing to conventional dose chemotherapy.

Pharmacodynamic biomarkers in metronomic chemotherapy: multiplex cytokine measurements in gastrointestinal cancer patients.

Metronomic chemotherapy has shown promising antitumor activity in a number of malignancies. We previously reported a phase II clinical trial of metronomic UFT (a 5-fluorouracil prodrug; 100 mg/twice per day p.o.) and cyclophosphamide (CTX; 500 mg/m2 i.v. bolus on day 1 and then 50 mg/day p.o.) plus celecoxib (200 mg/twice a day p.o.) in 38 patients with advanced refractory gastrointestinal tumors. The mechanisms of action of metronomic chemotherapy include inhibition of angiogenesis, direct cytotoxic effects on cancer cells, and, at least for drugs such as CTX, activation of the immune system. To further evaluate the latter, we carried out an immune system multiplex 14-cytokine profiling of plasma samples that were available (for day 0, day 28, and day 56) from 31 of the 38 patients in the above-noted clinical trial. Our results show that pre-treatment plasma-level cutoffs of interferon gamma (> 12.84 pg/ml), sCD40L (< 2168 pg/ml), interferon alpha 2 (> 55.11 pg/ml), and IL-17a (< 15.1 pg/ml) were predictive markers for those patients with better progression-free survival (p < .05 for each cytokine). After 28 days of metronomic therapy, the plasma levels of sCD40L, IL-17a, and IL-6 (< 130 pg/ml) could serve as predictors of improved progression-free survival, as could levels interferon gamma and sCD40L after 56 days of therapy. We observed minimal changes in cytokine profiles, from baseline, as a consequence of the metronomic therapy, with the exception of an elevation of IL-6 and IL-8 levels 28 days (and 56 days) after treatment started (p < 0.05). Our results indicate that a selective cytokine elevation involves IL-6 and IL-8, following metronomic chemotherapy administration. In addition, interferon gamma and sCD40L may be potential biomarkers for gastrointestinal cancer patients that are likely to benefit from metronomic chemotherapy. Our study contributes to our understanding of the mechanisms of action of metronomic chemotherapy, and the cytokine profiling we describe may guide future selection of gastrointestinal cancer patients for UFT/CTX/celecoxib combination metronomic chemotherapy.

Synergistic activity of linifanib and irinotecan increases the survival of mice bearing orthotopically implanted human anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer, and novel combined therapies are urgently needed to prolong patient survival. No data are currently available on the preclinical activity of the combination of linifanib, a CSF-1R inhibitor, and irinotecan in ATC. The aim of the study was to evaluate the in vitro and in vivo activity of linifanib plus irinotecan. Proliferation and apoptosis assays were performed on 8305C and 8505C human ATC cell lines exposed to SN-38, the active metabolite of irinotecan, linifanib alone, and their concomitant combination. Synergism was evaluated by the combination index method. Quantification of pospho-CSF-1R levels was performed by ELISA. In vivo ATC orthotopic xenografts were treated with the single drugs, or their combination, to evaluate their impact on survival. Histology and immunohistochemistry were performed on ATC tissue samples. Both SN-38 and linifanib inhibited in vitro the proliferation of 8305C and 8505C cells in a concentration-dependent manner, whereas their concomitant treatment revealed a strong synergism in the ATC cells. A significant pro-apoptotic activity was found in both ATC cell lines treated with linifanib alone and in combination with SN-38. Moreover, linifanib significantly decreased the levels of phospho-CSF-1R after 24 h and 72 h in both 8505C and 8305C cells, and this was also observed with the concomitant administration of SN-38. In vivo, the combination of linifanib and irinotecan produced a greater survival result than either monotherapy, and resulted in a significant higher median survival. In some of the mice the combination produced a complete response with a macroscopic disappearance of the disease, as confirmed by histology. In conclusion, the synergistic ATC antitumor activity of linifanib/irinotecan combination significantly increased the survival of ATC affected mice and induced some complete responses, suggesting a potential role of this schedule in ATC patient's treatment.

Development of a VLP-Based Vaccine Displaying an xCT Extracellular Domain for the Treatment of Metastatic Breast Cancer.

Metastatic breast cancer (MBC) is the leading cause of cancer death in women due to recurrence and resistance to conventional therapies. Thus, MBC represents an important unmet clinical need for new treatments. In this paper we generated a virus-like particle (VLP)-based vaccine (AX09) to inhibit de novo metastasis formation and ultimately prolong the survival of patients with MBC. To this aim, we engineered the bacteriophage MS2 VLP to display an extracellular loop of xCT, a promising therapeutic target involved in tumor progression and metastasis formation. Elevated levels of this protein are observed in a high percentage of invasive mammary ductal tumors including triple negative breast cancer (TNBC) and correlate with poor overall survival. Moreover, xCT expression is restricted to only a few normal cell types. Here, we tested AX09 in several MBC mouse models and showed that it was well-tolerated and elicited a strong antibody response against xCT. This antibody-based response resulted in the inhibition of xCT's function in vitro and reduced metastasis formation in vivo. Thus, AX09 represents a promising novel approach for MBC, and it is currently advancing to clinical development.

Pharmacological effects of vinorelbine in combination with lenvatinib in anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is a rare neoplasia with a poor prognosis. Proliferation and apoptosis assays were performed on ATC cell lines (8305C, 8505C) exposed to vinorelbine, lenvatinib, as well as to concomitant combinations. ABCB1, ABCG2 and CSF-1 mRNA expression was evaluated by real time PCR. The relative levels of pospho Akt were investigated as part of a human phospho-kinase array analysis, and CSF-1 and VEGFR-2 protein levels were measured by ELISA. The intracellular concentration of lenvatinib in ATC cells was measured by combined reversed-phase liquid chromatography-tandem mass spectrometry. An ATC subcutaneous xenograft tumor model in nude mice was treated with vinorelbine, lenvatinib, or vinorelbine plus lenvatinib. After treatment with vinorelbine, lenvatinib, a significant antiproliferative effect in ATC cell lines was observed. The concomitant treatment of vinorelbine and lenvatinib revealed synergism for all the fractions of affected cells. A decrease in ABCB1 expression was reported in both ATC cell lines treated with the lenvatinib plus vinorelbine combination, as was an increase in the intracellular concentration of lenvatinib. The combination caused a decrease in Akt, GSK3α/ß, PRAS40 and Src phosphorylation, and in both CSF-1 mRNA and protein levels. In the subcutaneous tumor model, the combination reduced the tumor volume during the treatment period. Our results establish the synergistic ATC antitumor activity of a vinorelbine and lenvatinib combination.

Overexpression of p62/IMP2 can Promote Cell Migration in Hepatocellular Carcinoma via Activation of the Wnt/ß-Catenin Pathway.

p62/IMP2 is an oncofetal protein that was first reported as a tumor-associated antigen in hepatocellular carcinoma (HCC). In our previous studies, we demonstrated a high frequency of p62/IMP2 autoantibodies appearing in various types of cancer. Therefore, we hypothesize that p62/IMP2 plays an important role in the progression of HCC, although the mechanism remains to be explored. In this study, we evaluated the expression of p62/IMP2 protein both in human tissues and liver cancer cell lines by immunohistochemistry and western blotting analysis and found that p62/IMP2 protein is overexpressed in human HCC tissue in comparison to normal human liver tissue. To explore the role that p62/IMP2 plays in HCC, p62/IMP2 was knocked out in two p62/IMP2-positive liver cancer cell lines (SNU449 and HepG2). Due to the low expression level of p62/IMP2 in SNU449, we overexpressed p62/IMP2 in this cell line. We subsequently demonstrated that high expression of p62/IMP2 in both cell lines can promote cell migration and invasion abilities in vitro by activating the Wnt/ß-catenin pathway. We also used the Wnt/ß-catenin pathway inhibitor, XAV 939, and a phosphoproteome assay to confirm our findings. Conclusion: Our results suggest that p62/IMP2 is an essential regulator of Wnt signaling pathways and plays an important role in HCC progression and metastasis.

Optimization of biomarkers-based classification scores as progression-free survival predictors: an intuitive graphical representation.

Aim: To construct classification scores based on a combination of cancer patient plasma biomarker levels, for predicting progression-free survival. Methods: The approach is based on the optimization of the biomarker cut-off values, which maximize the statistical differences between the groups with values lower or larger than the cut-offs, respectively. An intuitive visualization of the quality of the classification score is also proposed. Results: Even if there are only weak correlations between individual biomarker levels and progression-free survival, scores based on suitably chosen combination of three biomarkers have classification power comparable with the Response Evaluation Criteria in Solid Tumors criteria classification of response to treatments in solid tumors. Conclusion: Our approach has the potential to improve the selection of the patients who will benefit from a given anticancer treatment.

Precision therapeutic targeting of human cancer cell motility.

Increased cancer cell motility constitutes a root cause of end organ destruction and mortality, but its complex regulation represents a barrier to precision targeting. We use the unique characteristics of small molecules to probe and selectively modulate cell motility. By coupling efficient chemical synthesis routes to multiple upfront in parallel phenotypic screens, we identify that KBU2046 inhibits cell motility and cell invasion in vitro. Across three different murine models of human prostate and breast cancer, KBU2046 inhibits metastasis, decreases bone destruction, and prolongs survival at nanomolar blood concentrations after oral administration. Comprehensive molecular, cellular and systemic-level assays all support a high level of selectivity. KBU2046 binds chaperone heterocomplexes, selectively alters binding of client proteins that regulate motility, and lacks all the hallmarks of classical chaperone inhibitors, including toxicity. We identify a unique cell motility regulatory mechanism and synthesize a targeted therapeutic, providing a platform to pursue studies in humans.

Spatiotemporal assessment of spontaneous metastasis formation using multimodal in vivo imaging in HER2+ and triple negative metastatic breast cancer xenograft models in mice.

BACKGROUND: Preclinical breast cancer models recapitulating the clinical course of metastatic disease are crucial for drug development. Highly metastatic cell lines forming spontaneous metastasis following orthotopic implantation were previously developed and characterized regarding their biological and histological characteristics. This study aimed to non-invasively and longitudinally characterize the spatiotemporal pattern of metastasis formation and progression in the MDA-MB-231-derived triple negative LM2-4 and HER2+ LM2-4H2N cell lines, using bioluminescence imaging (BLI), contrast enhanced computed tomography (CT), fluorescence imaging, and 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography ([18F]FDG-PET). MATERIAL AND METHODS: LM2-4, LM2-4H2N, and MDA-MB-231 tumors were established in the right inguinal mammary fat pad (MFP) of female SCID mice and resected 14-16 days later. Metastasis formation was monitored using BLI. Metabolic activity of primary and metastatic lesions in mice bearing LM2-4 or LM2-4H2N was assessed by [18F]FDG-PET. Metastatic burden at study endpoint was assessed by CT and fluorescence imaging following intravenous dual-modality liposome agent administration. RESULTS: Comparable temporal metastasis patterns were observed using BLI for the highly metastatic cell lines LM2-4 and LM2-4H2N, while metastasis formed about 10 days later for MDA-MB-231. 21 days post primary tumor resection, metastases were detected in 86% of LM2-4, 69% of LM2-4H2N, and 60% of MDA-MB-231 inoculated mice, predominantly in the axillary region, contralateral MFP, and liver/lung. LM2-4 and LM2-4H2N tumors displayed high metabolism based on [18F]FDG-PET uptake. Lung metastases were detected as the [18F]FDG-PET uptake increased significantly between pre- and post-metastasis scan. Using a liposomal dual-modality agent, CT and fluorescence confirmed BLI detected lesions and identified additional metastatic nodules in the intraperitoneal cavity and lung. CONCLUSIONS: The combination of complementary anatomical and functional imaging techniques can provide high sensitivity characterization of metastatic disease spread, progression and overall disease burden. The described models and imaging toolset can be implemented as an effective means for quantitative treatment response evaluation in metastatic breast cancer.

Metronomic vinorelbine is directly active on Non Small Cell Lung Cancer cells and sensitizes the EGFRL858R/T790M cells to reversible EGFR tyrosine kinase inhibitors.

Metronomic vinorelbine (mVNR) has been described primarily as an antiangiogenic therapy, and no direct effects of mVNR on Non Small Cell Lung Cancer (NSCLC) cells has yet been demonstrated. The aims of this study were i) to establish the direct activity of mVNR on NSCLC cells either EGFR wt or EGFRL858R/T790M, and ii) to quantify the synergism of the combination with reversible EGFR tyrosine kinase inhibitors (TKIs), investigating the underlying mechanism of action. Proliferation assays were performed on A-549 (wt EGFRhigh), H-292 (EGFR-wt), H-358 (EGFR-wt), H-1975 (EGFRL858R/T790M) NSCLC cell lines exposed to mVNR, its active metabolite deacetyl-VNR (D-VNR), gefitinib and erlotinib for 144 h treatments. The synergism between mVNR and EGFR TKIs was determined by the combination index (CI) in EGFR-wt and H-1975 NSCLC cells. Cyclin-D1 and ABCG2 genes expression and protein levels were measured by RT-PCR and ELISA assays, as well as the phosphorylation of ERK1/2 and Akt. Intracellular concentrations of EGFR TKIs and VNR were investigated with a mass spectrometry system. mVNR, and its active metabolite D-VNR, were extremely active on NSCLC cells, in particular on H-1975 (IC50 = 13.56 ±â€¯2.77 pM), resistant to TKIs. mVNR inhibited the phosphorylation of ERK1/2 and Akt and significantly decreased the expression of both cyclin-D1 and ABCG2 m-RNA and protein. The simultaneous combination of VNR and reversible EGFR TKIs showed a strong synergism on EGFR-wt NSCLC cells and on H-1975 cells (e.g. CI = 0.501 for 50% of affected cells), increasing the intracellular concentrations of EGFR TKIs (e.g. +50.5% vs. gefitinib alone). In conclusions, mVNR has direct effects on NSCLC cells and sensitizes resistant cells to EGFR TKIs, increasing their intracellular concentrations.

Bromoethylindole (BEI-9) redirects NF-κB signaling induced by camptothecin and TNFα to promote cell death in colon cancer cells.

Chemotherapeutic regimens containing camptothecin (CPT), 5-fluorouracil, and oxaliplatin are used to treat advanced colorectal cancer. We previously reported that an indole derivative, 3-(2-bromoethyl)indole (BEI-9), inhibited the proliferation of colon cancer cells and suppressed NF-κB activation. Here, we show that a combination of BEI-9 with either CPT or tumor necrosis factor alpha (TNFα) enhances cell death. Using colorectal cancer cells, we examined the activation of NF-κB by drugs, the potential of BEI-9 for inhibiting drug-induced NF-κB activation, and the enhancement of cell death by combination treatments. Cells were treated with the chemotherapeutic drugs alone or in combination with BEI-9. NF-κB activation, cell cycle profiles, DNA-damage response, markers of cell death signaling and targets of NF-κB were evaluated to determine the effects of single and co-treatments. The combination of BEI-9 with CPT or TNFα inhibited NF-κB activation and reduced the expression of NF-κB-responsive genes, Bcl-xL and COX2. Compared to CPT or BEI-9 alone, sequential treatment of the cells with CPT and BEI-9 significantly enhanced caspase activation and cell death. Co-treatment with TNFα and BEI-9 also caused more cytotoxicity than TNFα or BEI-9 alone. Combined BEI-9 and TNFα enhanced cell death through caspase activation and cleavage of the switch-protein, RIP1 kinase. BEI-9 reduced the expression of COX2 both alone and in combination with CPT or TNF. We postulate that BEI-9 enhances the effects of these drugs on cancer cells by turning off or redirecting NF-κB signaling. Therefore, the combination of BEI-9 with drugs that activate NF-κB needs to be evaluated for clinical applications.

Synergistic efficacy of irinotecan and sunitinib combination in preclinical models of anaplastic thyroid cancer.

The identification of new therapeutic strategies is urgently needed for the management of patients affected by anaplastic thyroid cancer (ATC) due to their short survival and poor prognosis. Aim of the study was to determine the activity of the combination irinotecan/sunitinib on ATC cell growth in vitro and the antitumor effects in vivo. Proliferation assays were performed for 72 h on ATC cell lines exposed to the combination of SN-38, the active metabolite of irinotecan, and sunitinib. The simultaneous combination of sunitinib and SN-38, quantified by the combination index, determined a high synergism on ATC cells, increasing the intracellular concentrations of SN-38. Moreover, the synergistic combination greatly decreases the gene expression and the protein levels of vascular endothelial growth factor, colony stimulating factor 1 and ATP-binding cassette transporter G2 in ATC cells. A significant in vivo antitumor effect was observed in ATC xenografts with the simultaneous combination of irinotecan and sunitinib if compared to monotherapy. The simultaneous combination of irinotecan and sunitinib, in vitro and in vivo demonstrated a significant, synergistic ATC antitumor activity, suggesting a possible and rapid translation of this schedule into the clinics.

Resistance to metronomic chemotherapy and ways to overcome it.

Therapeutic resistance is amongst the major determinants of cancer mortality. Contrary to initial expectations, antivascular therapies are equally prone to inherent or acquired resistance as other cancer treatment modalities. However, studies into resistance to vascular endothelial growth factor pathway inhibitors revealed distinct mechanisms of resistance compared to conventional cytotoxic therapy. While some of these novel mechanisms of resistance also appear to be functional regarding metronomic chemotherapy, herein we summarize available evidence for mechanisms of resistance specifically described in the context of metronomic chemotherapy. Numerous preclinically identified molecular targets and pathways represent promising avenues to overcome resistance and enhance the benefits achieved with metronomic chemotherapy eventually. However, there are considerable challenges to clinically translate the preclinical findings.

Impact of CTLA-4 blockade in conjunction with metronomic chemotherapy on preclinical breast cancer growth.

BACKGROUND: Although there are reports that metronomic cyclophosphamide (CTX) can be immune stimulating, the impact of its combination with anti-CTLA-4 immunotherapy for the treatment of cancer remains to be evaluated. METHODS: Murine EMT-6/P breast cancer, or its cisplatin or CTX-resistant variants, or CT-26 colon, were implanted into Balb/c mice. Established tumours were monitored for relative growth following treatment with anti-CTLA-4 antibody alone or in combination with; (a) metronomic CTX (ldCTX; 20 mg kg-1 day-1), b) bolus (150 mg kg-1) plus ldCTX, or (c) sequential treatment with gemcitabine (160 mg kg-1 every 3 days). RESULTS: EMT-6/P tumours responded to anti-CTLA-4 therapy, but this response was less effective when combined with bolus plus ldCTX. Anti-CTLA-4 could be effectively combined with either ldCTX (without a bolus), or with regimens of either sequential or concomitant gemcitabine, including in orthotopic EMT-6 tumours, and independently of the schedule of drug administration. Tumour responses were confirmed with CT-26 tumours but were less pronounced in drug-resistant EMT-6/CTX or EMT-6/DDP tumour models than in the parent tumour. A number of tumour bearing mice developed spontaneous metastases under continuous therapy. The majority of cured mice rejected tumour re-challenges. CONCLUSIONS: Metronomic CTX can be combined with anti-CTLA-4 therapy, but this therapy is impaired by concomitant bolus CTX. Sequential therapy of anti-CTLA-4 followed by gemcitabine is effective in chemotherapy-naive tumours, although tumour relapses can occur, in some cases accompanied by the development of spontaneous metastases.

Label-Free Raman Imaging to Monitor Breast Tumor Signatures.

Although not yet ready for clinical application, methods based on Raman spectroscopy have shown significant potential in identifying, characterizing, and discriminating between noncancerous and cancerous specimens. Real-time and accurate medical diagnosis achievable through this vibrational optical method largely benefits from improvements in current technological and software capabilities. Not only is the acquisition of spectral information now possible in milliseconds and analysis of hundreds of thousands of data points achieved in minutes, but Raman spectroscopy also allows simultaneous detection and monitoring of several biological components. Besides demonstrating a significant Raman signature distinction between nontumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, our study demonstrates that Raman can be used as a label-free method to evaluate epidermal growth factor activity in tumor cells. Comparative Raman profiles and images of specimens in the presence or absence of epidermal growth factor show important differences in regions attributed to lipid, protein, and nucleic acid vibrations. The occurrence, which is dependent on the presence of epidermal growth factor, of new Raman features associated with the appearance of phosphothreonine and phosphoserine residues reflects a signal transduction from the membrane to the nucleus, with concomitant modification of DNA/RNA structural characteristics. Parallel Western blotting analysis reveals an epidermal growth factor induction of phosphorylated Akt protein, corroborating the Raman results. The analysis presented in this work is an important step toward Raman-based evaluation of biological activity of epidermal growth factor receptors on the surfaces of breast cancer cells. With the ultimate future goal of clinically implementing Raman-guided techniques for the diagnosis of breast tumors (e.g., with regard to specific receptor activity), the current results just lay the foundation for further label-free optical tools to diagnose the disease.

Clinical, pharmacodynamic and pharmacokinetic results of a prospective phase II study on oral metronomic vinorelbine and dexamethasone in castration-resistant prostate cancer patients.

The aim of the present study was to evaluate clinical activity, and the pharmacodynamic and pharmacokinetic profiles, of oral metronomic vinorelbine (VNR) plus dexamethasone (DEX) in metastatic castration-resistant prostate cancer (mCRPC) patients. Fourty-one patients (92 % chemotherapy-resistant) received 30 mg/day VNR p.o. thrice a week plus 1 mg/day DEX p.o. until disease progression. Plasma soluble B cell antigen 7 homolog 3 (sB7-H3), vascular endothelial growth factor (VEGF), and thrombospondin-1 (TSP-1), were measured by ELISA. Plasma VNR was detected using a LC-MS-MS system. The fraction of patients free of progression, defined by criteria of the Prostate Cancer Clinical Trials Working Group 2, at 3 months was 61 %. PSA decrease ≥50 % from baseline was observed in 35 % of patients. Median PFS and OS were 4 months (95 % CI, 2.8-6.9) and 17.5 months (95 % CI, 10.8-24.5), respectively. Toxicity was mild, and no grade 4 toxicities were found. The mean plasma VNR Cmax ranged from 1 to 2.7 ng/ml (Tmax 1.1 h) and no evidence of drug accumulation was found. A moderate relationship was found between plasma sB7-H3 and PSA values (r = 0.565; P = 0.0094) at the baseline. Increased PFS (11.3 vs. 2.8 months; P = 0.0298) was observed in patients with sB7-H3 levels <30.25 ng/mL. Plasma VEGF AUC0-24day increased in non-responders (P < 0.0001), whereas responders maintained higher plasma TSP-1 AUC0-24day (P = 0.0063). In conclusion, metronomic VNR plus DEX showed favourable activity, and a low toxicity profile, in mCRPC patients. Plasma sB7-H3, VEGF and TSP-1 levels are potential pharmacodynamic markers at the reached low plasma concentrations of vinorelbine metronomically administered.

A novel curcumin-like dienone induces apoptosis in triple-negative breast cancer cells.

PURPOSE: According to the World Health Organization (WHO), breast cancer is the most common cancer affecting women worldwide. In the USA ~12.3 % of all women are expected to be diagnosed with various types of breast cancer, exhibiting varying degrees of therapeutic response rates. Therefore, the identification of novel anti-breast cancer drugs is of paramount importance. METHODS: The 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore was incorporated into a number of cytotoxins. Three of the resulting dienones, 2a, 2b and 2c, were tested for their anti-neoplastic potencies in a variety of human breast cancer-derived cell lines, including the triple negative MDA-MB-231 cell line and its metastatic variant, using a live-cell bio-imaging method. Special emphasis was put on dienone 2c, since its anti-cancer activity and its mode of inflicting cell death have so far not been reported. RESULTS: We found that all three dienones exhibited potent cytotoxicities towards the breast cancer-derived cell lines tested, whereas significantly lower toxicities were observed towards the non-cancerous human breast cell line MCF-10A. The dienones 2b and 2c exhibited the greatest selective cytotoxicity at submicromolar concentration levels. We found that these two dienones induced phosphatidylserine externalization in MDA-MB-231 cells in a concentration-dependent manner, suggesting that their cytotoxic effect might be mediated by apoptosis. This possibility was confirmed by our observation that the dienone 2c can induce mitochondrial depolarization, caspase-3 activation, cell cycle disruption and DNA fragmentation in MDA-MB-231 cells. CONCLUSION: Our findings indicate that dienone 2c uses the mitochondrial/intrinsic pathway to inflict apoptosis in triple negative MDA-MB-231 breast cancer-derived cells. This observation warrants further assessment of dienone 2c as a potential anti-breast cancer drug.