Chemo-Cryo Combination Therapy: An Adjunctive Model for the Treatment of Prostate Cancer

Despite continuing research and the development of alternate therapeutic options, prostate cancer remains problematic. Chemotherapy has played a minor role as a treatment option due to its lack of efficacy. Whereas cryotherapy has received renewed attention as a treatment modality, it too fails to offer an absolute curative option. Previously, we reported on the utilization of a therapeutic model, which, in combination, increases cell death in a canine renal cell model. Based upon that study, we investigated a combination therapy model as an alternative for the treatment modality for prostate cancer. We hypothesized that the combination of chemotherapy and cryosurgery would result in enhanced cell death, thereby presenting a more effective treatment of prostate cancer. A human prostate cancer cell (PC-3) model was exposed to 5-fluorouracil (5-FU) for 2 and 4 days (prefreeze), freezing (-5 to -100 degrees C), or a combination of the two treatments, and each was assessed for effectiveness over a 2-week posttreatment period. Additionally, investigation into the mechanisms of cell death initiated by the respective therapies was performed through DNA cleavage analysis. For chemotherapy, cultures exposed to 5-FU (2-4 days) yielded a 15-25% loss in cell survival. For cryotherapy, cultures exposed to a temperature window of -5 to -20 degrees C yielded an initial 5-70% loss of viability but cells propagated over time. Cultures exposed to temperatures of -25 to -80 degrees C yielded a 90-99% (+/-4.5%) initial loss in viability with repopulation observed by 12 days postthaw. Cells frozen to -100 degrees C yielded 100% (+/-0.3%) loss of viability and exhibited no signs of propagation. For chemo-cryo therapy, combination treatment at milder temperatures (-5 to -25 degrees C) resulted in an enhanced loss of cell viability compared to that for either treatment alone. Combination treatment at lower temperatures (-40 to -80 degrees C) resulted in a complete loss of cell viability. DNA fragmentation analysis at 48 h posttreatment revealed that dead (detached) cells treated with 5-FU died primarily through apoptosis, whereas dead cells from freezing (-15 degrees C) alone died primarily through freeze-rupture and necrosis. Detached cell analysis from combination treatment at -15 degrees C revealed the presence of apoptotic, necrotic, and freeze-rupture cell death. Scanning electron micrographs of cells exposed to freezing contributing to cell death. These data demonstrate that the combination of 5-FU at sublethal doses and freezing temperatures improves human prostate cancer cell death efficacy. Further, we suggest that chemo-cryo therapy offers a potential alternative treatment for the control and eradication of prostate cancer.     

Death receptor-induced cell death in prostate cancer

Prostate cancer mortality results from metastasis and is often coupled with progression from androgen-dependent to androgen-independent growth. Unfortunately, no effective treatment for metastatic prostate cancer increasing patient survival is available. The absence of effective therapies reflects in part a lack of knowledge about the molecular mechanisms involved in the development and progression of this disease. Apoptosis, or programmed cell death, is a cell suicide mechanism that enables multicellular organisms to regulate cell number in tissues. Inhibition of apoptosis appears to be a critical pathophysiological factor contributing to the development and progression of prostate cancer. Understanding the mechanism(s) of apoptosis inhibition may be the basis for developing more effective therapeutic approaches. Our understanding of apoptosis in prostate cancer is relatively limited when compared to other malignancies, in particular, hematopoietic tumors. Thus, a clear need for a better understanding of apoptosis in this malignancy remains. In this review we have focused on what is known about apoptosis in prostate cancer and, more specifically, the receptor/ligand-mediated pathways of apoptosis as potential therapeutic targets.     

Using extracellular biomarkers for monitoring efficacy of therapeutics in cancer patients: an update

Rapidly detectable and easily accessible markers of tumor cell death are needed for evaluating early therapeutic efficacy for immunotherapy and chemotherapy so that patients and their physicians can decide whether to remain with a given therapeutic strategy. Currently, image-based tests such as computed tomography scans and magnetic resonance imaging are used to visualize the response of a patient’s tumor, but often these evaluations are not conducted for weeks to months after treatment begins. While serum levels of secreted proteins such as carcinoembryonic antigen and prostate specific antigen are commonly monitored to gauge tumor status during therapy and between image evaluations, the levels of these proteins do not always correlate well with the actual tumor response. In laboratory studies, it has been shown that tumor cells undergoing apoptosis can release cellular components into cell culture media such as cytochrome c, nucleosomes, cleaved cytokeratin-18 and E-cadherin. Studies of patient sera have found that these and other macromolecules can be found in circulation during cancer therapy, providing a potential source of material for monitoring treatment efficacy. In the future, analysis of biofluids from severe combined immunodeficiency mice bearing patient tumor specimens treated with a targeted therapy such as Apo2L/tumor necrosis factor-related apoptosis-inducing ligand will be useful in the preclinical identification of therapy response markers. In this review, the current status of the identification of serum markers of tumor cell apoptosis is provided, as well as a discussion of critical research questions that must be addressed and the considerations necessary when identifying a marker that reflects true clinical outcome.     

Gene therapy in cancer

Gene therapy, recently frequently investigated, is an alternative treatment method that introduces therapeutic genes into a cancer cell or tissue to cause cell death or slow down the growth of the cancer. This treatment has various strategies such as therapeutic gene activation or silencing of unwanted or defective genes; therefore a wide variety of genes and viral or nonviral vectors are being used in studies. Gene therapy strategies in cancer can be classified as inhibition of oncogene activation, activation of tumor suppressor gene, immunotherapy, suicide gene therapy and antiangiogenic gene therapy. In this review, we explain gene therapy, gene therapy strategies in cancer, approved gene medicines for cancer treatment and future of gene therapy in cancer. Today gene therapy has not yet reached the level of replacing conventional therapies. However, with a better understanding of the mechanism of cancer to determine the right treatment and target, in the future gene therapy, used as monotherapy or in combination with another existing treatment options, is likely to be used as a new medical procedure that will make cancer a controllable disease.     

Dual inhibition of autophagy and the AKT pathway in prostate cancer

Genetic inactivation of PTEN through either gene deletion or mutation is common in metastatic prostate cancer, leading to activation of the phosphoinositide 3-kinase (PI3K-AKT) pathway, which is associated with poor clinical outcomes. The PI3K-AKT pathway plays a central role in various cellular processes supporting cell growth and survival of tumor cells. To date, therapeutic approaches to develop inhibitors targeting the PI3K-AKT pathway have failed in both pre-clinical and clinical trials. We showed that a novel AKT inhibitor, AZD5363, inhibits the AKT downstream pathway by reducing p-MTOR and p-RPS6KB/p70S6K. We specifically reported that AZD5363 monotherapy induces G2 growth arrest and autophagy, but fails to induce significant apoptosis in PC-3 and DU145 prostate cancer cell lines. Blocking autophagy using pharmacological inhibitors (3-methyladenine, chloroquine and bafilomycin A₁) or genetic inhibitors (siRNA targeting ATG3 and ATG7) enhances cell death induced by AZD5363 in these prostate cancer cells. Importantly, the combination of AZD5363 with chloroquine significantly reduces tumor volume compared with the control group, and compared with either drug alone in prostate tumor xenograft models. Taken together, these data demonstrate that AKT inhibitor AZD5363, synergizes with the lysosomotropic inhibitor of autophagy, chloroquine, to induce apoptosis and delay tumor progression in prostate cancer models that are resistant to monotherapy, with AZD5363 providing a new therapeutic approach potentially translatable to patients.     

Regulated lytic cell death in breast cancer

Breast cancer (BC) is a very common cancer among women and one of the primary causes of death in women worldwide. Because BC has different molecular subtypes, the challenges associated with targeted therapy have increased significantly, and the identification of new therapeutic targets has become increasingly urgent. Blocking apoptosis and inhibiting cell death are important characteristics of malignant tumours, including BC. Under adverse conditions, including exposure to antitumour therapy, inhibition of cell death programmes can promote cancerous transformation and the survival of cancer cells. Therefore, inducing cell death in cancer cells is fundamentally important and provides new opportunities for potential therapeutic interventions. Lytic forms of cell death, primarily pyroptosis, necroptosis and ferroptosis, are different from apoptosis owing to their characteristic lysis, that is, the production of cellular components, to guide beneficial immune responses, and the application of lytic cell death (LCD) in the field of tumour therapy has attracted considerable interest from researchers. The latest clinical research results confirm that lytic death signalling cascades involve the BC cell immune response and resistance to therapies used in clinical practice. In this review, we discuss the current knowledge regarding the various forms of LCD, placing a special emphasis on signalling pathways and their implications in BC, which may facilitate the development of novel and optimal strategies for the clinical treatment of BC.     

Effective and new potent drug combination: Histone deacetylase and Wnt/β-catenin pathway inhibitors in lung carcinoma cells

Lung cancer is the most commonly diagnosed cancer worldwide with a high mortality rate. In this study, the therapeutic effect of combination valproic acid and niclosamide was investigated on human lung cancer cell line. The effects of the compounds alone and combination therapy on cell viability were determined by sulforhodamine B and adenosine 5′-triphosphate viability assays. Flow cytometry was used to determine the cell death mechanism and DNA damage levels responsible for the cytotoxic effects of combination therapy. The presence of apoptosis in cells was supported by fluorescence microscopy and also by using inhibitors of the apoptotic signaling pathway. The increase in cellular reactive oxygen species (ROS) level in combination therapy was determined by H2DCFDA staining. The effect of N-acetyl-l -cysteine combination on ROS increase was investigated on cell viability. In addition, the expression levels of the proteins associated with epigenetic regulation and cell death were analyzed by Western blotting and gene expression levels were determined using real-time quantitative polymerase chain reaction.It was observed that the combination therapy showed a cytotoxic effect on the A549 lung cancer cells compared to the individual use of the inhibitors. The absence of this effect on normal lung cells revealed the presence of a selective toxic effect. When the mechanism of cytotoxicity is examined, it has been observed that combination therapy initiates the activation of tumor necrosis receptors and causes apoptosis by activated caspase. It was also observed that this extrinsic apoptotic pathway was activated on the mitochondrial pathway. In addition, ER stress and mitochondrial membrane potential loss associated with increased ROS levels induce cell death. When the data in this study were evaluated, combination therapy caused a dramatic decrease in cell viability by inducing the extrinsic apoptotic pathway in lung cancer cell line. Therefore, it was concluded that it can be used as an effective and new treatment option for lung cancer.     

Pretreatment of docetaxel enhances TRAIL-mediated apoptosis in prostate cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In this study, we examined whether treatment of docetaxel (DTX) can enhance apoptotic cell death by TRAIL against androgen-independent prostate cancer (AIPC). The cell death effect of combinations of TRAIL and docetaxel on prostate cancer cell lines (androgen-dependent LNCaP and its derived androgen-independent, metastatic C4-2B) was evaluated by synergisms of apoptosis. Western blot assay and DNA fragmentation assay were used to study the underlying mechanisms of cell death and search for any mechanisms of enhancement of TRAIL induced apoptosis in the presence of docetaxel. In addition, we investigated the in vitro anti-tumor effects of combined docetaxel and TRAIL using MAP kinase inhibitors. Docetaxel itself could not induce apoptotic cell death in 24 h even in high concentration. Apoptotic cell death, however, was drastically enhanced by pretreatment of docetaxel 20 h before TRAIL treatment. Docetaxel enhanced the PARP-1 cleavage and caspases activation by TRAIL especially in androgen-independent, metastatic C4-2B cell line, mainly by phosphorylation of Bcl-2 by JNK activation. It appears that apoptotic cell death was protected by the JNK inhibitor SP600125. The results of our study show that pretreatment of docetaxel is able to enhance the apoptosis produced by TRAIL in prostate cancer cells, especially in hormone-refractory prostate cancer (HRPC).