PI3K/Akt/mTOR pathway inhibitors enhance radiosensitivity in radioresistant prostate cancer cells through inducing apoptosis, reducing autophagy, suppressing NHEJ and HR repair pathways.

The PI3K/Akt/mTOR pathway has a central role in cancer metastasis and radiotherapy. To develop effective therapeutics to improve radiosensitivity, understanding the possible pathways of radioresistance involved and the effects of a combination of the PI3K/Akt/mTOR inhibitors with radiotherapy on prostate cancer (CaP) radioresistant cells is needed. We found that compared with parent CaP cells, CaP-radioresistant cells demonstrated G0/G1 and S phase arrest, activation of cell cycle check point, autophagy and DNA repair pathway proteins, and inactivation of apoptotic proteins. We also demonstrated that compared with combination of single PI3K or mTOR inhibitors (BKM120 or Rapamycin) and radiation, low-dose of dual PI3K/mTOR inhibitors (BEZ235 or PI103) combined with radiation greatly improved treatment efficacy by repressing colony formation, inducing more apoptosis, leading to the arrest of the G2/M phase, increased double-strand break levels and less inactivation of cell cycle check point, autophagy and non-homologous end joining (NHEJ)/homologous recombination (HR) repair pathway proteins in CaP-radioresistant cells. This study describes the possible pathways associated with CaP radioresistance and demonstrates the putative mechanisms of the radiosensitization effect in CaP-resistant cells in the combination treatment. The findings from this study suggest that the combination of dual PI3K/Akt/mTOR inhibitors (BEZ235 or PI103) with radiotherapy is a promising modality for the treatment of CaP to overcome radioresistance.

Acquisition of epithelial-mesenchymal transition and cancer stem cell phenotypes is associated with activation of the PI3K/Akt/mTOR pathway in prostate cancer radioresistance.

Radioresistance is a major challenge in prostate cancer (CaP) radiotherapy (RT). In this study, we investigated the role and association of epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and the PI3K/Akt/mTOR signaling pathway in CaP radioresistance. We developed three novel CaP radioresistant (RR) cell lines (PC-3RR, DU145RR and LNCaPRR) by radiation treatment and confirmed their radioresistance using a clonogenic survival assay. Compared with untreated CaP-control cells, the CaP-RR cells had increased colony formation, invasion ability and spheroid formation capability (P<0.05). In addition, enhanced EMT/CSC phenotypes and activation of the checkpoint proteins (Chk1 and Chk2) and the PI3K/Akt/mTOR signaling pathway proteins were also found in CaP-RR cells using immunofluorescence, western blotting and quantitative real-time PCR (qRT-PCR). Furthermore, combination of a dual PI3K/mTOR inhibitor (BEZ235) with RT effectively increased radiosensitivity and induced more apoptosis in CaP-RR cells, concomitantly correlated with the reduced expression of EMT/CSC markers and the PI3K/Akt/mTOR signaling pathway proteins compared with RT alone. Our findings indicate that CaP radioresistance is associated with EMT and enhanced CSC phenotypes via activation of the PI3K/Akt/mTOR signaling pathway, and that the combination of BEZ235 with RT is a promising modality to overcome radioresistance in the treatment of CaP. This combination approach warrants future in vivo animal study and clinical trials.

Co-expression of CD147 (EMMPRIN), CD44v3-10, MDR1 and monocarboxylate transporters is associated with prostate cancer drug resistance and progression.

BACKGROUND: The aim of this study is to seek an association between markers of metastatic potential, drug resistance-related protein and monocarboxylate transporters in prostate cancer (CaP). METHODS: We evaluated the expression of invasive markers (CD147, CD44v3-10), drug-resistance protein (MDR1) and monocarboxylate transporters (MCT1 and MCT4) in CaP metastatic cell lines and CaP tissue microarrays (n=140) by immunostaining. The co-expression of CD147 and CD44v3-10 with that of MDR1, MCT1 and MCT4 in CaP cell lines was evaluated using confocal microscopy. The relationship between the expression of CD147 and CD44v3-10 and the sensitivity (IC(50)) to docetaxel in CaP cell lines was assessed using MTT assay. The relationship between expression of CD44v3-10, MDR1 and MCT4 and various clinicopathological CaP progression parameters was examined. RESULTS: CD147 and CD44v3-10 were co-expressed with MDR1, MCT1 and MCT4 in primary and metastatic CaP cells. Both CD147 and CD44v3-10 expression levels were inversely related to docetaxel sensitivity (IC(50)) in metastatic CaP cell lines. Overexpression of CD44v3-10, MDR1 and MCT4 was found in most primary CaP tissues, and was significantly associated with CaP progression. CONCLUSIONS: Our results suggest that the overexpression of CD147, CD44v3-10, MDR1 and MCT4 is associated with CaP progression. Expression of both CD147 and CD44v3-10 is correlated with drug resistance during CaP metastasis and could be a useful potential therapeutic target in advanced disease.

CD147/EMMPRIN and CD44 are potential therapeutic targets for metastatic prostate cancer.

Prostate cancer (CaP) is a major health problem in males in Western countries. Current therapeutic approaches are limited and many patients die of secondary disease (metastases). There is no cure for metastatic castration-resistant prostate cancer (CRPC). Targeting tumor-associated antigens is fast emerging as an area of promise to treat late stage and recurrent CaP. Extracellular matrix metalloproteinase inducer, EMMPRIN (CD147) is a multifunctional glycoprotein that can modify the tumor microenvironment by activating proteinases, inducing angiogenic factors in tumor and stromal cells, and regulating growth and survival of anchorage-independent tumor cells (micrometastases) and multidrug resistance (MDR). CD44 is a multifunctional protein involved in cell adhesion, migration and drug resistance, and is a primary receptor for hyaluronan (HA), a major component of the extracellular matrix (ECM) with a critical role in cell signaling and cell-ECM interactions in cancer. Our recent studies indicate both CD147 and CD44 are involved in cancer drug resistance and play very important roles in CaP metastasis. Thus, CD147 and CD44 may be ideal therapeutic targets to control metastatic and CRPC disease. This review will discuss their putative roles in CaP metastasis and MDR, and give an overview of literature regarding their expression on human CaP tissues. Additional focus will be on the potential of therapeutic strategies targeting CD147 and CD44 to prevent CaP metastasis and overcome drug resistance.

Targeting uPA/uPAR in prostate cancer.

Prostate cancer (CaP) is one of the most common malignancies in men, with an increasing incidence. Despite significant advances in surgery, chemotherapy and radiotherapy to treat CaP, many patients unfortunately succumb to secondary disease (metastases). The invasive ability of tumour cells plays a key role in CaP metastasis and is a major cause of treatment failure. Urokinase plasminogen activator (uPA) and its receptor (uPAR)-mediated signalling have been implicated in tumour cell invasion, survival, and metastasis in a variety of cancers including CaP. Both uPA and uPAR are expressed at much higher levels in CaP tissues than in benign and normal prostate tissues. They are used as diagnostic markers as well as therapeutic targets due to their aberrant and unique expression pattern during cancer progression. Current therapeutic options for patients with metastatic hormone-refractory CaP (HRPC) are very limited. Therefore, much effort is currently being directed toward targeting aberrant uPA or uPAR activity in CaP. This review summarizes some important new findings supporting the role of uPA/uPAR in CaP progression and establishing the potential therapeutic efficacy of uPA/uPAR-targeted therapies in CaP.

MUC1 is a promising therapeutic target for prostate cancer therapy.

Prostate cancer (CaP) is one of the most common malignancies in men, and the incidence of CaP is increasing. Because of the limitations of current therapeutic approaches, many patients die of secondary disease (metastases). Mucins are used as diagnostic markers as well as therapeutic targets due to their aberrant and unique expression pattern during cancer progression. There is a growing interest in mucins as treatment targets in human malignancies, including CaP. So far, 21 mucin genes have been identified. Of these, MUC1 has been investigated most extensively. In neoplastic tissues, MUC1 is underglycosylated compared with that in normal tissues. The reduced glycosylation permits the immune system to access the peptide core of the tumor-associated underglycosylated MUC1 antigen (uMUC1) and reveal epitopes that are masked in the normal cell. This feature makes it possible to design an antibody that discriminates between normal and adenocarcinoma cells and target tumor-associated MUC1 with toxins or radionuclides, or use a vaccine targeting tumor-associated MUC1 antigen. The results from our recent study have shown that over-expression of MUC1 plays a very important role in CaP progression and MUC1 is an ideal target for targeted therapy to control micrometastases and hormone refractory disease. This review will cover our current understanding of the structure and functions of MUC1, summarize its expression on human CaP tissues and focus on the MUC1-based immunotherapy for control of metastatic CaP.

Immunohistochemical characterisation of the monoclonal antibody BLCA-38 for the detection of prostate cancer.

BACKGROUND: Monoclonal antibodies (MAbs) can be used to detect, image and treat cancers. This study aimed to characterise the binding of BLCA-38 MAbs to human prostate cancer cell lines, human prostate cancer biopsy samples and normal tissues to enable future targeted studies. METHODS: BLCA-38 antigen expression on cancer lines was determined by flow cytometry; that on patient specimens from normal tissues and cancers was tested by immunohistochemistry using fresh frozen tissues or paraffin-embedded tissues that had undergone antigen retrieval. RESULTS: Cell surface BLCA-38 antigen expression was seen on DU-145, PC-3, PC-3 M and PC-3 M-MM2 prostate cancer lines, but LNCaP, MDA PCa 2a or MDA PCa 2b lines were negative. Other human lines, including 8/12 bladder cancer and A431 vulval epidermoid cells, but not breast cancer lines, expressed BLCA-38 antigen. Staining occurred in glandular epithelial cells in the majority of frozen, and paraffin-embedded prostate cancer tissues and was occasionally seen in prostatic intraepithelial neoplasia (PIN). No staining was observed in normal cadaver tissues or in benign areas from various other cancer tissues. CONCLUSIONS: The BLCA-38 antibody binds to the majority of human prostate cancers but not to normal cells, and has potential for targeting novel therapies in patients with this disease.

Toxicology and pharmacokinetics of intravesical gemcitabine: a preclinical study in dogs.

More active and well-tolerated agents are needed for the treatment of superficial bladder cancer. This study investigated intravesical gemcitabine to establish the toxicology and pharmacokinetics necessary for clinical trials. Beagle dogs (in groups of 2; n = 6) received 100 mg, 350 mg, or 1 g of drug by intravesical administration on alternate days three times/week for 4 weeks. Animals were observed for clinical signs of toxicity; gemcitabine levels and peripheral blood counts were taken three times weekly. The dogs were euthanized, and a full necropsy was performed at days 1 and 14 after the last dose. Intravesical gemcitabine was given at 100 mg (n = 2), 350 mg (equivalent to the 1000 mg/m2 human dose; n = 3), and 3.5 g (n = 1). i.v. gemcitabine was given at 350 mg (n = 2). Plasma samples drawn at time points up to 8 h were analyzed for systemic absorption and clearance of drug. Doses of 100 and 350 mg were well tolerated with no clinical side effects. Necropsies revealed normal bone marrow cellularity and normal bladder histology. At 1 g, signs of severe clinical toxicity were evident, and after only three doses, necropsies demonstrated severe bone marrow hypoplasia, cystitis, and intestinal necrosis. At all intravesical doses, significant systemic absorption was seen. The T1/2 (+/- SD) for intravesical and i.v. administration of 350 mg was 328 (+/-6.8) min and 99.3 (+/-5.2) min, respectively (P<0.001). Intravesical gemcitabine is well tolerated and has no direct bladder toxicity at doses up to 1000 mg/m2. Higher doses result in gastrointestinal, bladder, and bone marrow toxicity.

A fellowship in the USA: essential information for the Australasian medical graduate.

BACKGROUND: There have been many recent changes to the immigration and educational requirements necessary to obtain both clinical and research positions in the USA. This paper describes these requirements in detail and examines the cost of obtaining a position. METHODS: An extensive review of Medline and relevant documents from educational and immigration bodies is undertaken in addition to personal and peer experience of the processes necessary to fulfil educational and immigration requirements for funded medical positions in the USA. A cost analysis is performed based upon the latest charges for exam requirements and visa fees. A 'minimum' cost for fulfilling exam and visa requirements only and a 'likely' cost (which represents a more realistic appraisal of the expenses likely to be incurred) are investigated. RESULTS: This review shows that many recent changes have made the attainment of a position difficult and expensive. Much foresight and planning is required to obtain the necessary requirements for medical licensure and to obtain working visa status in addition to the application for the position itself. The cost analysis reveals that the minimum cost is $4050 (AU$) and the 'likely' cost is $8600 (AU$). CONCLUSION: Australasian trainees and Fellows need to be fully aware of the difficulties and expenses likely to be encountered in order to make an informed decision with regard to overseas training in the USA.

Cryotherapy for neuroendocrine liver metastases.

While the prognosis for patients with untreated liver metastases from neuroendocrine primaries is rather good, they often suffer disabling symptoms due to syndromes of hormonal excess. Thirteen patients with metastatic neuroendocrine tumours were treated by hepatic cryotherapy; seven patients were symptomatic and five of these had elevated levels of hormonal tumour markers. Twelve patients are alive and mostly asymptomatic with a median follow up of 13.5 months; one patient died after 45 months of bronchopneumonia without evidence of tumour recurrence. All patients with elevated preoperative tumour markers have had a significant fall in markers postoperatively. Two patients were returned to the operating theatre for coagulopathy-associated bleeding: one patient each developed acute renal failure and pulmonary embolism, but there was no mortality. This study shows that hepatic cryotherapy offers a useful treatment option for this group of patients, alleviates symptoms and may have an impact on survival.

Renal cryotherapy in a sheep model; a feasibility study.

PURPOSE: This study was designed to evaluate the safety and efficacy of renal cryotherapy as a possible treatment of renal malignancy with preservation of renal parenchyma. MATERIALS AND METHODS: Ten Merino sheep were anaesthetised and the right kidney was exposed through a retro-peritoneal approach. A 5 mm. cryotherapy probe (LCS 3000 Cryotec UK) was inserted into the lower pole of the kidney and freezing was undertaken to form an iceball 5 cm. in diameter. RESULTS: There was no mortality and no complications were observed. A transient rise in creatinine was observed post-operatively. The sheep were euthenased at 4 weeks and at necropsy macroscopic examination revealed a contracted, fibrotic wedge shaped lesion of 3 cm in diameter. Histological examination of the "cryolesion" revealed a central area of coagulative necrosis and a 5 mm rim of partial necrosis with preservation of renal tubules. CONCLUSION: We conclude that renal cryotherapy is safe and can achieve effective renal necrosis in the sheep model.

Cryosurgery.

The in situ destruction of malignant tissue by freezing is a relatively new addition to the list of treatment options for patients with cancer in solid organs. Although skin cancers have long been treated by cryosurgery, the lack of suitable equipment for both delivering and monitoring the freezing process hindered further development of the treatment of more inaccessible lesions until the 1960s. At this time, Cooperz developed cryosurgical machines which enabled the destruction of CNS lesions in patients with Parkinson's disease and stimulated renewed interest in the application of cryosurgery to the treatment of cancer.

In vitro and in vivo assessment of urethral warming catheters for the transperineal cryoablation of prostatic carcinoma.

OBJECTIVE: To evaluate the two most common urethral warming catheters currently available for their effectiveness in preventing urethral injury in an animal model and thus during transperineal cryosurgery for the treatment of prostate cancer. PATIENTS, MATERIALS AND METHODS: The warming efficiency of the variable-diameter thin latex-balloon catheter (Cook, Australia) and the fixed-diameter PVC catheter (Candela, USA) were evaluated in a water-bath. A sheep model was then used for subsequent experiments to further evaluate the better catheter. The technique used for the insertion of the Cook catheter in a series of 33 patients treated with prostate cryosurgery is also described. RESULTS: In vitro, the Cook catheter was more effective (P < 0.01) than the Candela catheter in preventing localized cooling. In vivo, results in five animals showed that the urethral epithelium was viable in all sections that had been protected by the catheter and focally denuded or necrotic in those sections not protected, with each animal used as its own control (P < 0.01 Fisher's exact test). The 33 patients treated while using the Cook catheter had no subsequent complications. CONCLUSION: The urethral warming catheter currently in use for prostatic cryosurgery in Australia is more effective in vitro than its competitor and is effective in preventing urethral injury in vivo.

Two cases of spontaneous liver rupture and literature review.

Spontaneous liver rupture is uncommon, difficult to diagnose and carries a universally high mortality. It has been well documented to occur as a complication of primary or secondary hepatic malignancy. Similarly, there are 28 cases of ruptured haemangiomata described in the world literature. It is also well described in severe pregnancy-induced hypertension and is said to carry a mortality of 18% for patients treated by packing and drainage of the haematoma and 75% for patients treated with liver resection. Two female patients aged 60 and 61 presented to our accident and emergency department. One had a history of hypertension only and the other a history of a bleeding diathesis from the lupus anticoagulant. Both presented with hypotension and abdominal pain and both were diagnosed by abdominal CT scan. One was treated with hepatic artery ligation and tamponade and the other with liver resection and correction of the coagulopathy. Neither had any evidence of a ruptured hemangioma or tumor at laparotomy or on histological examination, and both are alive and well. The conclusions to be drawn from this review and our own recent experience is that the treatment of choice for ruptured haemangiomata is liver resection and, for rupture during pregnancy, is tamponade with packs and evacuation of the haematoma. Hepatic arteriography and embolisation, if possible, is a useful adjunct. Correction of any coagulopathy is essential. We can only speculate that the aetiology in our patients was uncontrolled hypertension in one and coagulopathy in the other.

Laparoscopic vs open ultrasound of the liver: an in vitro study.

Intra-operative contact ultrasound is a sensitive method of detecting liver tumours. The aim of this study was to compare the sensitivity of open contact ultrasound (OUS) of the liver with laparoscopic contact ultrasound (LUS). Hypoechoic "lesions" were created in 5 fresh pig livers by inserting 28 grapes via small incisions in the inferior surface. The size (range 8-25 mm) and location of each grape was recorded. Scanning was undertaken in random order by two experienced independent observers with no knowledge of the size, number or position of the lesions, using an Aloka 650 series scanner and 7.5 MHz probes. The crude sensitivity with OUS was 96% and 100% respectively for the two observers, and 92% for each with LUS. One grape was interpreted as 2 separate grapes on LUS by one observer. Absolute sensitivity (grapes identified in the correct location) was 86% and 93% respectively with OUS and 79% for each observer with LUS. LUS was almost as sensitive as OUS in this model of hepatic metastases.

Critical temperature for in vivo cryoablation of human prostate cancer in a xenograft model.

OBJECTIVES: To assess the in vivo sensitivity of human prostate cancer cells (PC3) to cryoablation and thus define the minimum temperature needed to prevent the recurrence of cancer after percutaneous transperineal radical prostatic cryoablation. MATERIALS AND METHODS: Twenty-five male nu/nu mice were inoculated by a subcutaneous injection with 5 x 10(5) PC3 cells. After 3 weeks the tumours (mean area 60.4 mm2, SEM 5.7) were frozen using a 3 mm cryotherapy probe (LCS 3000 Cryotech UK) to temperatures ranging from 0 degrees to -40 degrees C. RESULTS: No tumours recurred in seven mice which had tumours frozen to < -15 degrees C. There were eight recurrences in 15 mice which had tumours frozen to between 0 degree and -15 degrees C, and all were confirmed histologically. CONCLUSIONS: The minimum temperature required to prevent recurrence of tumours from human prostate cancer cells was < -15 degrees C in this in vivo mouse model.

Bioactivity of recombinant feline interleukin-2 on human and feline leukocytes.

Interleukin (IL)-2 is a 16,000 Da protein product of T lymphocytes which is the principle cytokine responsible for clonal expansion of T lymphocytes as a response to antigen exposure. Deficiency of functional IL-2 plays a pivotal role in the pathogenesis of human immunodeficiency syndrome and may be important in the pathogenesis of feline immunodeficiency syndrome as well. Additionally, IL-2 may enhance secretion of interleukin-5 from the TH2 subset of CD4+ T cells, promote peripheral and systemic eosinophilia, and contribute to the eosinophilia which characterizes the inflamed airways of human beings and cats with asthma. We recently reported the sequence of feline IL-2 and the synthesis of recombinant feline IL-2. The purpose of the present study was to evaluate the bioactivity of recombinant feline IL-2 on human and feline leukocytes. We established dose-response relationships between recombinant feline IL-2 and radiolabeled proliferating human and feline leukocytes using thymidine incorporation as a marker of bioactivity. We found that recombinant human IL-2 promotes proliferation of both human and feline leukocytes. However, recombinant feline IL-2 promotes proliferation of feline cells, but not human cells.

Cryotherapy treatment of patients with hepatic metastases from neuroendocrine tumors.

BACKGROUND: Liver metastases from neuroendocrine tumors often present with disabling symptoms due to syndromes of hormonal excess. A locally destructive technique such as hepatic cryotherapy not only alleviates symptoms but may improve survival in this group of patients. METHODS: Six patients with metastatic neuroendocrine tumors were treated with hepatic cryotherapy. Four patients were symptomatic and three of these had elevated tumor markers from ectopic hormone production. RESULTS: All patients are alive and asymptomatic, with a median follow-up of 24 months (range, 6 months to 6 years). All have had a complete radiologic response. All with elevated preoperative markers have had a greater than 89% decrease in tumor markers. Coagulopathy occurred in two patients necessitating additional surgery, but there was no other morbidity attributable to the cryotherapy. CONCLUSION: To the authors' knowledge, this study demonstrates for the first time that hepatic cryotherapy offers supportive treatment for patients with neuroendocrine tumors metastatic to the liver. Cryotherapy alleviates symptoms and may improve survival.