Emerging roles of radioresistance in prostate cancer metastasis and radiation therapy.

Radiation therapy (RT) continues to be one of the most popular treatment options for localized prostate cancer (CaP). Local CaP recurrence after RT is a pattern of treatment failure attributable to radioresistance of cancer cells. One major obstacle to RT is that there is a limit to the amount of radiation that can be safely delivered to the target organ. Recent results indicate that phosphoinositide 3-kinase (PI3K)/Akt/phosphatase and tensin homolog (PTEN)/mammalian target of rapamycin (mTOR) signaling pathway, autophagy, epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) are involved in CaP metastasis and radioresistance. Emerging evidence also suggests that combining a radiosensitizer with RT increases the efficacy of CaP treatment. Understanding the mechanisms of radioresistance will help to overcome recurrence after RT in CaP patients and prevent metastasis. In this review, we discuss the novel findings of PI3K/Akt/PTEN/mTOR signaling pathway, autophagy, EMT and CSCs in the regulation of CaP metastasis and radioresistance, and focus on combination of radiosensitizers with RT in the treatment of CaP in preclinical studies to explore novel approaches for future clinical trials.

A preliminary study to develop an intervention to facilitate communication between couples in advanced cancer.

OBJECTIVE: Psychosocial interventions directed to couples where one has advanced cancer can reduce distress, enhance communication, and provide an opportunity for relational growth. The present study aimed to develop an intervention to facilitate communication about living with advanced cancer using the Patient Dignity Inventory (PDI) as the focus of a clinical interview with couples toward the end of life. METHOD: Couples were recruited from oncology and palliative care services at a Sydney hospital. After the PDI was developed and manualized as an intervention for couples, the PDI-Couple Interview (PDI-CI) was delivered by a clinical psychologist and comprised the following: (1) the patient completed the PDI; (2) the patient's identified partner completed the PDI about how they thought the patient was feeling; and (3) the clinician reviewed the results with the couple, summarizing areas of concurrence and discordance and facilitating discussion. RESULTS: Some 34 couples were referred, of which 12 consented, 9 of whom completed the clinical interview. Reported benefits included enabling couples to express their concerns together, identifying differences in understanding, and giving "permission to speak" with each other. The focus of the interview around the PDI provided a structure that was particularly acceptable for men. Most couples confirmed that they were "on the same page," and where differences were identified, it provided a forum for discussion and a mutual understanding of the challenges in managing advanced cancer within a supportive context. SIGNIFICANCE OF RESULTS: Participant couples' experiences of the PDI-CI provide valuable insight into the benefits of this intervention. This preliminary study indicates that the intervention is a relatively simple means of enhancing closer communication and connection between couples where one has advanced cancer and may be an important adjunct in helping prepare couples for the challenges inherent toward the end of life. Further investigation of feasibility with a larger sample is recommended.

CD44 variant 6 is associated with prostate cancer metastasis and chemo-/radioresistance.

INTRODUCTION: Prostate cancer (CaP) is the second leading malignancy in older men in Western countries. The role of CD44 variant 6 (CD44v6) in CaP progression and therapeutic resistance is still uncertain. Here, we investigated the roles of CD44v6 in CaP metastasis and chemo/radioresistance. Expression of CD44v6 in metastatic CaP cell lines, human primary CaP tissues and lymph node metastases was assessed using immunofluorescence and immunohistochemistry, respectively. METHODS: Knock down (KD) of CD44v6 was performed in PC-3M, DU145, and LNCaP cells using small interfering RNA (siRNA), and confirmed by confocal microscope, Western blot and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The adhesive ability and invasive potential were assessed using a hyaluronic acid (HA) adhesion and a matrigel chamber assay, respectively. Tumorigenesis potential and chemo-/radiosensitivity were measured by a sphere formation assay and a colony assay, respectively. RESULTS: Over-expression of CD44v6 was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of CD44v6 suppressed CaP proliferative, invasive and adhesive abilities, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated epithelial-mesenchymal transition (EMT), PI3K/Akt/mTOR, and Wnt/ß-catenin signaling pathway proteins in vitro. CONCLUSIONS: Our findings demonstrate that CD44v6 is an important cancer stem cell-like marker associated with CaP proliferation, invasion, adhesion, metastasis, chemo-/radioresistance, and the induction of EMT as well as the activation PI3K/Akt/mTOR and Wnt signaling pathways, suggesting that CD44v6 is a novel therapeutic target to sensitize CaP cells to chemo/radiotherapy.

'Workshops in healing' for senior medical students: a 5-year overview and appraisal.

We report upon the design, content and feedback from an interactive, experiential series of Workshops in Healing for senior medical students. Fifty-six final year medical students enrolled in 2×3 h workshops designed around the core themes of 'physician know thyself' (Workshop 1) and 'confronting suffering' (Workshop 2). Of the 56 students who initially enrolled, 48 students completed both workshops and provided a written open-ended reflection of their learning experience. The study, undertaken over a consecutive 5-year period (2008-2012), employed an emergent, qualitative design using thematic analysis of the reflective comments. We found that the design and content of both workshops promoted transformative learning for these final year medical students. Students identified the following benefits: (1) the opportunity to reaffirm their commitment to their chosen career path; (2) the value of listening to other students share their stories; (3) the importance of the timing of the workshops to occur after exams; (4) the use of various mediums such as art, poetry, music and contemporary/classic literature to present concepts of suffering and healing; and (5) the creation of a safe and confidential space. Students reported that these innovative workshops gave them a renewed sense of drive and enthusiasm for their chosen career. They highlighted the importance of addressing an aspect of medicine (healing) not covered in the traditional medical curriculum. Workshops in Healing helped them to rediscover a deeper meaning to medicine and their roles as future healthcare professionals.

The impact of breast cosmetic and functional outcomes on quality of life: long-term results from the St. George and Wollongong randomized breast boost trial.

The aims of this study were to evaluate the impact of cosmetic and functional outcomes after breast-conserving surgery (BCS) and radiation on quality of life (QOL). In this exploratory analysis; baseline, 5 and 10 years data of patient's assessment of breast cosmesis, arm swelling/pain, limitation of movement, loss of feeling in fingers and breast sensitivity/tenderness were dichotomized and their impact on QOL (QLQ-C30) were assessed. Multivariable modelling was also performed to assess associations with QOL. The St. George and Wollongong randomized trial randomized 688 patients into the boost and no boost arms. 609, 580, and 428 patients had baseline, 5 and 10 years cosmetic data available, respectively. Similar numbers had the various functional assessments in the corresponding period. By univariate analysis, cosmesis and a number of functional outcomes were highly associated with QOL. Adjusted multivariate modelling showed that cosmesis remained associated with QOL at 5 and 10 years. Breast sensitivity, arm pain, breast separation, age and any distant cancer event were also associated with QOL on multivariate modelling at 10 years. This study highlights the importance of maintaining favorable cosmetic and functional outcomes following BCS. In addition, the clinically and statistically significant relationship between functional outcomes and QOL shows the importance for clinicians and allied health professionals in identifying, discussing, managing, and limiting these effects in women with breast cancer in order to maintain QOL.

Proteomics for breast cancer urine biomarkers.

Although the survival of breast cancer (BC) patients has increased over the last two decades due to improved screening programs and postoperative adjuvant systemic therapies, many patients die from metastatic relapse. Current biomarkers used in the clinic are not useful for the early detection of BC, or monitoring its progression, and have limited value in predicting response to treatment. The development of proteomic techniques has sparked new searches for novel protein markers for many diseases including BC. Proteomic techniques allow for a high-throughput analysis of samples with the visualization and quantification of thousands of potential protein and peptide markers. Human urine is one of the most interesting and useful biofluids for routine testing and provides an excellent resource for the discovery of novel biomarkers, with the advantage over tissue biopsy samples due to the ease and less invasive nature of collection. In this review, we summarize the results from studies where urine was used as a source for BC biomarker research and discuss urine sample preparation, its advantage, challenges, and limitation. We focus on the gel-based proteomic approaches as well as the recent development of quantitative techniques in BC urine biomarker detection. Finally, the future use of modern proteomic techniques in BC biomarker identification will be discussed.

Low dose histone deacetylase inhibitor, LBH589, potentiates anticancer effect of docetaxel in epithelial ovarian cancer via PI3K/Akt pathway in vitro.

The purpose of this study was to investigate the effect of combination of LBH589 with docetaxel (DTX) on the growth and survival of epithelial ovarian cancer (EOC) cells in vitro and the possible mechanisms of chemo-sensitization of LBH589 in the combination treatment. The effect of LBH589 alone or in combination with DTX on four EOC cell lines (OVCAR-3, IGROV-1, A2780 and SKOV-3) was studied by MTT and clonogenic assays, acridine orange (AO)/ethidium bromide (EB) staining for apoptosis, Western blotting for apoptosis-related proteins, histone H3 and H4 proteins, DNA double strand break (DSB) repair marker and phosphorylation of Akt. LBH589 alone inhibited EOC cell proliferation in a time and dose-dependent manner. Low-dose of LBH589 (IC(20)) combined with DTX had an additive effect and greatly improved efficacy of DTX cell killing in EOC cells. Compared to DTX alone, the combination treatment with LBH589 and DTX induced more apoptosis and led to an increased and persistent DSB. Cell death following single or combined treatment was associated with the release of cytochrome c activity, increased caspase-3 (active) and PARP-1(cleaved), histone acetylation-related proteins and PI3k/Akt signaling pathway. Our results suggest that LBH589 enhances DTX-induced apoptosis in human EOC cells, and can be used in combination with DTX as an attractive strategy for treating human EOC.

Combination therapy with the histone deacetylase inhibitor LBH589 and radiation is an effective regimen for prostate cancer cells.

Radiation therapy (RT) continues to be one of the most popular treatment options for localized prostate cancer (CaP). The purpose of the study was to investigate the in vitro effect of LBH589 alone and in combination with RT on the growth and survival of CaP cell lines and the possible mechanisms of radiosensitization of this combination therapy. The effect of LBH589 alone or in combination with RT on two CaP cell lines (PC-3 and LNCaP) and a normal prostatic epithelial cell line (RWPE-1) was studied by MTT and clonogenic assays, cell cycle analysis, western blotting of apoptosis-related and cell check point proteins, and DNA double strand break (DSB) repair markers. The immunofluorescence staining was used to further confirm DSB expression in treated CaP cells. Our results indicate that LBH589 inhibited proliferation in both CaP and normal prostatic epithelial cells in a time-and-dose-dependent manner; low-dose of LBH589 (IC20) combined with RT greatly improved efficiency of cell killing in CaP cells; compared to RT alone, the combination treatment with LBH589 and RT induced more apoptosis and led to a steady increase of sub-G1 population and abolishment of RT-induced G2/M arrest, increased and persistent DSB, less activation of non-homologous end joining (NHEJ)/homologous recombination (HR) repair pathways and a panel of cell cycle related proteins. These results suggest that LBH589 is a potential agent to increase radiosensitivity of human CaP cells. LBH589 used either alone, or in combination with RT is an attractive strategy for treating human CaP.

Tear fluid protein biomarkers.

The tear film covers and protects the ocular surface. It contains various molecules including a large variety of proteins. The protein composition of the tear fluid can change with respect to various local and systemic diseases. Prior to the advent of the proteomic era, tear protein analysis was limited to a few analytical techniques, the most common of which was immunoelectrophoresis, an approach dependent on antibody availability. Using proteomics, hundreds of tear proteins could potentially be identified and subsequently studied. Although detection of low-abundance proteins in the complex tear proteome remains a challenge, advances in sample fractionation and mass spectrometry have greatly enhanced our ability to detect these proteins. With increasing proteomic applications, tears show great potential as biomarkers in the development of clinical assays for various human diseases. In this chapter, we discuss the structure and functions of the tear film and methods for its collection. We also summarize potential tear protein biomarkers identified using proteomic techniques for both ocular and systemic diseases. Finally, modern proteomic techniques for tear biomarker research and future challenges are explored.

"Tu Souffres, Cela Suffit": the compassionate hospital.

The authors propose that the characteristics of personal (individual) compassion may be extrapolated to the concept of corporate (organizational) compassion. Modern health care facilities attract staff members who are able to exercise varying degrees of compassion in their busy daily routines. However, little discussion has taken place on how health care organizations might best harness and integrate aspects of individual compassion to create an organization with compassion as a core value. We define three characteristics of a "compassionate hospital" as 1) the presence of a healing environment, 2) a sense of connection among people, and 3) a sense of purpose and identity. We suggest how a "top down" focus on compassion as a core value by clinical leaders could maximize the compassion of health care workers, and reduce the suffering expressed and/or experienced by health care workers and patients in today's health care facilities. The compassionate hospital concept is intended to act as a proposition for health policy researchers and decision-makers in health care so as to reduce the suffering of sick patients, and to restore a sense of well-being, meaning, and purpose among health care workers.

CD44 is a biomarker associated with human prostate cancer radiation sensitivity.

CD44 plays an important role in cancer metastasis, chemotherapy, and radiation resistance. The present study investigated the relationship of CD44 expression and radioresistance, and the potential mechanisms of CD44 in radiosensitivity using prostate cancer (CaP) cell lines. CD44 was knocked down in three CaP cell lines (PC-3, PC-3M-luc, and LNCaP) using small interfering RNA (siRNA) and clonogenic survival fractions after single dose irradiation were compared before and after CD44 knocking down (KD). The effect of radiation on cell cycle distribution was examined by flow cytometry and the cell cycle-related protein levels of phospho-Chk1 and phospho-Chk2 were ascertained by Western blotting. The expression of the DNA double strand break (DSB) marker-γH2AX was also quantified by immunofluorescence staining. Our results indicate that the down-regulation of CD44 enhanced radiosensitivity in PC-3, PC-3M-luc, and LNCaP CaP cells, the sensitizing enhancement ratio for these cell lines was 2.3, 1.3, and 1.5, respectively and that the delay of DNA DSB repair in low CD44-expressing KD CaP cells correlated with ineffective cell cycle arrest and the delayed phosphorylation of Chk1 and Chk2. These findings suggest that CD44 may be a valuable biomarker and a predictor of radiosensitivity in CaP treatment.

In vitro and in vivo prostate cancer metastasis and chemoresistance can be modulated by expression of either CD44 or CD147.

CD44 and CD147 are associated with cancer metastasis and progression. Our purpose in the study was to investigate the effects of down-regulation of CD44 or CD147 on the metastatic ability of prostate cancer (CaP) cells, their docetaxel (DTX) responsiveness and potential mechanisms involved in vitro and in vivo. CD44 and CD147 were knocked down (KD) in PC-3M-luc CaP cells using short hairpin RNA (shRNA). Expression of CD44, CD147, MRP2 (multi-drug resistance protein-2) and MCT4 (monocarboxylate tranporter-4) was evaluated using immunofluorescence and Western blotting. The DTX dose-response and proliferation was measured by MTT and colony assays, respectively. The invasive potential was assessed using a matrigel chamber assay. Signal transduction proteins in PI3K/Akt and MAPK/Erk pathways were assessed by Western blotting. An in vivo subcutaneous (s.c.) xenograft model was established to assess CaP tumorigenecity, lymph node metastases and DTX response. Our results indicated that KD of CD44 or CD147 decreased MCT4 and MRP2 expression, reduced CaP proliferation and invasive potential and enhanced DTX sensitivity; and KD of CD44 or CD147 down-regulated p-Akt and p-Erk, the main signal modulators associated with cell growth and survival. In vivo, CD44 or CD147-KD PC-3M-luc xenografts displayed suppressed tumor growth with increased DTX responsiveness compared to control xenografts. Both CD44 and CD147 enhance metastatic capacity and chemoresistance of CaP cells, potentially mediated by activation of the PI3K and MAPK pathways. Selective targeting of CD44/CD147 alone or combined with DTX may limit CaP metastasis and increase chemosensitivity, with promise for future CaP treatment.

Rembrandt, michelangelo, and stories of healing.

Two contemporary patient narratives provide an opportunity to reflect on the role of clinicians in the healing process, as seen in the works of Rembrandt and Michelangelo.

Anti-MUC1 monoclonal antibody (C595) and docetaxel markedly reduce tumor burden and ascites, and prolong survival in an in vivo ovarian cancer model.

MUC1 is associated with cellular transformation and tumorigenicity and is considered as an important tumor-associated antigen (TAA) for cancer therapy. We previously reported that anti-MUC1 monoclonal antibody C595 (MAb C595) plus docetaxel (DTX) increased efficacy of DTX alone and caused cultured human epithelial ovarian cancer (EOC) cells to undergo apoptosis. To further study the mechanisms of this combination-mediated apoptosis, we investigated the effectiveness of this combination therapy in vivo in an intraperitoneal (i.p.) EOC mouse model. OVCAR-3 cells were implanted intraperitoneally in female athymic nude mice and allowed to grow tumor and ascites. Mice were then treated with single MAb C595, DTX, combination test (MAb C595 and DTX), combination control (negative MAb IgG(3) and DTX) or vehicle control i.p. for 3 weeks. Treated mice were killed 4 weeks post-treatment. Ascites volume, tumor weight, CA125 levels from ascites and survival of animals were assessed. The expression of MUC1, CD31, Ki-67, TUNEL and apoptotic proteins in tumor xenografts was evaluated by immunohistochemistry. MAb C595 alone inhibited i.p. tumor growth and ascites production in a dose-dependent manner but did not obviously prevent tumor development. However, combination test significantly reduced ascites volume, tumor growth and metastases, CA125 levels in ascites and improved survival of treated mice compared with single agent-treated mice, combination control or vehicle control-treated mice (P<0.05). The data was in a good agreement with that from cultured cells in vitro. The mechanisms behind the observed effects could be through targeting MUC1 antigens, inhibition of tumor angiogenesis, and induction of apoptosis. Our results suggest that this combination approach can effectively reduce tumor burden and ascites, prolong survival of animals through induction of tumor apoptosis and necrosis, and may provide a potential therapy for advanced metastatic EOC.

Clinical pharmacology of isoflavones and its relevance for potential prevention of prostate cancer.

Isoflavones are phytoestrogens that have pleiotropic effects in a wide variety of cancer cell lines. Many of these biological effects involve key components of signal transduction pathways within cancer cells, including prostate cancer cells. Epidemiological studies have raised the hypothesis that isoflavones may play an important role in the prevention and modulation of prostate cancer growth. Since randomized phase III trials of isoflavones in prostate cancer prevention are currently lacking, the best evidence for this concept is presently provided by case control studies. However, in vitro data are much more convincing in regard to the activity of a number of isoflavones, and have led to the development of genistein and phenoxodiol in the clinic as potential treatments for cancer. In addition, the potential activity of isoflavones in combination with cytotoxics or radiotherapy warrants further investigation. This review focuses on the clinical pharmacology of isoflavones and its relevance to their development for use in the prevention of prostate cancer, and it evaluates some of the conflicting data in the literature.

Low levels of ATM in breast cancer patients with clinical radiosensitivity.

BACKGROUND AND PURPOSE: Adjuvant radiotherapy for cancer can result in severe adverse side effects for normal tissues. In this respect, individuals with anomalies of the ATM (ataxia telangiectasia) protein/gene are of particular interest as they may be at risk of both breast cancer and clinical radiosensitivity. The association of specific ATM gene mutations with these pathologies has been well documented, however, there is uncertainty regarding pathological thresholds for the ATM protein. RESULTS: Semi-quantitative immuno-blotting provided a reliable and reproducible method to compare levels of the ATM protein for a rare cohort of 20 cancer patients selected on the basis of their severe adverse normal tissue reactions to radiotherapy. We found that 4/12 (33%) of the breast cancer patients with severe adverse normal tissue reactions following radiotherapy had ATM protein levels < 55% compared to the mean for non-reactor controls. CONCLUSIONS: ATM mutations are generally considered low risk alleles for breast cancer and clinical radiosensitivity. From results reported here we propose a tentative ATM protein threshold of ~55% for high-risk of clinical radiosensitivity for breast cancer patients.

Prediction of local recurrence, distant metastases, and death after breast-conserving therapy in early-stage invasive breast cancer using a five-biomarker panel.

PURPOSE: To determine the clinical utility of intrinsic molecular phenotype after breast-conserving therapy (BCT) with lumpectomy and whole-breast irradiation with or without a cavity boost. PATIENTS AND METHODS: Four hundred ninety-eight patients with invasive breast cancer were enrolled into a randomized trial of BCT with or without a tumor bed radiation boost. Tumors were classified by intrinsic molecular phenotype as luminal A or B, HER-2, basal-like, or unclassified using a five-biomarker panel: estrogen receptor, progesterone receptor, HER-2, CK5/6, and epidermal growth factor receptor. Kaplan-Meier and Cox proportional hazards methodology were used to ascertain relationships to ipsilateral breast tumor recurrence (IBTR), locoregional recurrence (LRR), distant disease-free survival (DDFS), and death from breast cancer. RESULTS: Median follow-up was 84 months. Three hundred ninety-four patients were classified as luminal A, 23 were luminal B, 52 were basal, 13 were HER-2, and 16 were unclassified. There were 24 IBTR (4.8%), 35 LRR (7%), 47 distant metastases (9.4%), and 37 breast cancer deaths (7.4%). The overall 5-year disease-free rates for the whole cohort were: IBTR 97.4%, LRR 95.6%, DDFS 92.9%, and breast cancer-specific death 96.3%. A significant difference was observed for survival between subtypes for LRR (P = .012), DDFS (P = .0035), and breast cancer-specific death (P = .0482), but not for IBTR (P = .346). CONCLUSION: The 5-year and 10-year survival rates varied according to molecular subtype. Although this approach provides additional information to predict time to IBTR, LRR, DDFS, and death from breast cancer, its predictive power is less than that of traditional pathologic indices. This information may be useful in discussing outcomes and planning management with patients after BCT.

Preparation and testing of bevacizumab radioimmunoconjugates with Bismuth-213 and Bismuth-205/Bismuth-206.

Bevacizumab, a humanized anti-VEGF monoclonal antibody has shown promise in various clinical trials. We report the development and testing of Bi-213 (an alpha-emitting radionuclide) labeled bevacizumab for in vitro and in vivo studies using two different chelators viz cDTPA and CHX-A''. The developed labeling method showed high labeling yields of 93.6% and 89.7% for cDTPA and CHX-A'' respectively and the results were reproducible. The in vitro and in vivo stability tests were carried out using Bi-213 and long half-life Bismuth isotope (Bi-205/Bi-206) for pharmacokinetics. The in vitro results showed remarkable stability of the radiolabeled bevacizumab regardless of the chelator. The in vivo pharmacokinetics studies however, showed that the uptake and retention of cDTPA-bevacizumab was significantly higher in kidneys (p-value 0.02) and lower in liver and spleen (p-value <0.0001 and 0.0009 respectively). The values for the two conjugates compared well in blood but the longer term data for CHX-A'' conjugate showed slow clearance resulting in a significantly longer blood half-life of the product (211 hours compared to 4 hours for cDTPA-bevacizumab). Preliminary in vivo results showed increased efficacy of the combination therapy compared to bevacizumab only. The tumor area (mm(2)) decreased from 24.8 +/- 3.6 and 12.8 +/- 1.7 for 1 and 3 mg/kg cold bevacizumab only to 6.5 +/- 0.7 and 7.5 +/- 4.8 when single dose of 333 MBq/kg of (213)Bi-bevacizumab was administered as combination therapy. In conclusion it can be said that stable radiolabeled bevacizumab conjugates can be prepared with Bi-213 with either chelators used. The shorter blood half-life with cDTPA-bevacizumab may not be a major concern with Bi-213 as its own half-life is 46 minutes only. The combination therapy proved superior to bevacizumab alone therapy, a phenomenon that can be particularly useful in cancers where bevacizumab alone has shown limited success like prostate cancer.