Recent evidence suggests that the successful treatment of prostate cancer may require adjuvant therapies. Accordingly, a better understanding of the molecular mechanisms involved in current ...treatments may lead to enhanced efficacy by providing a basis for adjuvant therapies. In this study, we demonstrate that the combination of sub-lethal concentrations of chemotherapeutic agents prior to freezing (−15
°C) in a prostate cancer cell (PC-3) model results in enhanced efficacy over either treatment alone. Morphological analysis revealed that necrosis appeared to be the prevalent mode of cell death following adjuvant (in vitro) modeling, yet molecular analysis indicated that freezing and chemotherapy differentially activated apoptotic cascades through modulating opposing members of the Bcl-2 protein family. Freezing results in a time-dependent increase of the antiapoptotic Bcl-2 protein, while chemotherapy results in an increase of the pro-apoptotic Bax protein. Anti-apoptotic Bcl-2 protein levels increase over 3-fold following exposure to freezing. 5-Fluorouracil (5-FU) causes pro-apoptotic Bax levels to increase 2-fold during the drug exposure. The increase in Bax was also apparent following the combination of 5-FU/freezing, while Bcl-2 levels were maintained at or below control levels. This led to a shift in the Bcl-2 to Bax ratio to a pro-death tendency. Other effective cryo/chemo combinations were also found to provide similar effects. The combination of cisplatin/freezing resulted in a 4-fold increase in the ratio of Bax to Bcl-2 when compared to controls, which represented a 2-fold increase over the 5-FU/freezing-combination model. This increase may contribute to the continued reduction in cell number observed during the 13-day recovery period. Additionally, the addition of an apoptotic caspase inhibitor was not able to protect cultures from cell death following combination treatment. In conclusion, the data suggest that both Bcl-2 and Bax may, not only, play an important role in the efficacy of the cryo/chemo combination, but also the balance between the two may determine the role and extent of system destruction.
Tight junctions (TJs) play crucial roles in tissue homeostasis and inflammation through their roles in the control of paracellular transport and barrier function. There is evidence that these ...functions are compromised in older organisms, but the exact mechanisms leading to TJ deterioration are not well understood. Claudin proteins are a family of membrane proteins that constitute the structural barrier elements of TJs and therefore play a major role in their formation and function. Using immunohistochemistry and immunoblotting, we have studied the expression of six different claudin proteins (claudin-1, -2, -3, -4, -5, and -7) in three tissues (liver, kidney, and pancreas) of aging male and female mice. In general, we find an age-dependent decrease in the expression of several claudin proteins in all three tissues observed, although the exact changes are tissue specific. Our findings provide a possible basis for the decrease in tissue barrier function in older organisms.
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°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°C yielded an initial 5–70% loss of viability but cells propagated over time. Cultures exposed to temperatures of −25 to −80°C yielded a 90–99% (±4.5%) initial loss in viability with repopulation observed by 12 days postthaw. Cells frozen to −100°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°C) resulted in an enhanced loss of cell viability compared to that for either treatment alone. Combination treatment at lower temperatures (−40 to −80°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°C) alone died primarily through freeze-rupture and necrosis. Detached cell analysis from combination treatment at −15°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.
The techniques of present-day cryosurgery performed with multiprobe freezing apparatus and advanced imaging techniques yield predictable and encouraging results in the treatment of prostatic and ...renal cancers. Nevertheless, and not unique to cryosurgical treatment, the rates of persistent disease demonstrate the need for improvement in technique and emphasize the need for proper management of the therapeutic margin. The causes of persistent disease often relate to a range of factors including selection of patients, understanding of the extent of the tumor, limitations of the imaging techniques, and failure to freeze the tumor periphery in an efficacious manner. Of these diverse factors, the one most readily managed, but subject to therapeutic error, is the technique of freezing the tumor and appropriate margin to a lethal temperature Baust, J. G., Gage, A. A. The Molecular Basis of Cryosurgery. BJU Int 95, 1187–1191 (2005). This article describes the recent experiments that examine the molecular basis of cryosurgery, clarifies the actions of the components of the freeze-thaw cycle, and defines the resultant effect on the cryogenic lesion from a clinical perspective. Further, this review addresses the important issue of management of the margin of the tumor through adjunctive therapy. Accordingly, a goal of this review is to identify the technical and future adjunctive therapeutic practices that should improve the efficacy of cryoablative techniques for the treatment of malignant lesions.
Cryoablation has emerged as a primary therapy to treat prostate cancer. Although effective, the assumption that freezing serves as a ubiquitous lethal stress is challenged by clinical experience and ...experimental evidence demonstrating time-temperature-related cell-death dependence. The age-related transformation from an androgen-sensitive (AS) to an androgen-insensitive (AI) phenotype is a major challenge in the management of prostate cancer. AI cells exhibit morphological changes and treatment resistance to many therapies. As this resistance has been linked with alpha6beta4 integrin overexpression as a result of androgen receptor (AR) loss, we investigated whether alpha6beta4 integrin expression, as a result AR loss, contributes to the reported increased freeze tolerance of AI prostate cancer. A series of studies using AS (LNCaP LP and PC-3 AR) and AI (LNCaP HP and PC-3) cell lines were designed to investigate the cellular mechanisms contributing to variations in freezing response. Investigation into alpha6beta4 integrin expression revealed that AI cell lines overexpressed this protein, thereby altering morphological characteristics and increasing adhesion characteristics. Molecular investigations revealed a significant decrease in caspases-8, -9, and -3 levels in AI cells after freezing. Inhibition of alpha6beta4 integrin resulted in increased caspase activity after freezing (similar to AS cells) and enhanced cell death. These data show that AI cells show an increase in post-freeze susceptibility after inhibition of alpha6beta4 integrin function. Further understanding the role of androgen receptor-related alpha6beta4 integrin expression in prostate cancer cells responses to freezing might lead to novel options for neo-adjunctive treatments targeting the AR signaling pathway.
Cryosurgery offers a promising therapeutic alternative for the treatment of prostate cancer. While often successful, complete cryoablation of cancerous tissues sometimes fails due to technical ...challenges. Factors such as the end temperature, cooling rate, duration of the freezing episode, and repetition of the freezing cycle have been reported to influence cryosurgical outcome. Accordingly, we investigated the effects of these variables in an
in vitro prostate cancer model. Human prostate cancer PC-3 and LNCaP cultures were exposed to a range of sub-zero temperatures (−5 to −40
°C), and cells were thawed followed by return to 37
°C. Post-thaw viability was assessed using a variety of fluorescent probes including alamarBlue™ (metabolic activity), calceinAM (membrane integrity), and propidium iodide (necrosis). Freeze duration following ice nucleation was investigated using single and double freezing cycles (5, 10, and 20
min). The results demonstrated that lower freezing temperatures yielded greater cell death, and that LNCaP cells were more susceptible to freezing than PC-3 cells. At −15
°C, PC-3 yielded ∼55% viability versus ∼20% viability for LNCaP. Double freezing cycles were found to be more than twice as destructive versus a single freeze–thaw cycle. Both cell types experienced increased cell death when exposed to freezing temperatures for longer durations. When thawing rates were considered, passive (slower) thawing following freezing yielded greater cell death than active (faster) thawing. A 20% difference in viability between passive and active thawing was observed for PC-3 for a 10
min freeze. Finally, the results demonstrate that just reaching −40
°C
in vitro may not be sufficient to obtain complete cell death. The data support the use of extended freeze times, multiple freeze–thaw cycles, and passive thawing to provide maximum cell destruction.
The expanding complexity of biologics banked for therapeutic applications necessitates the development of improved preservation technologies for support of the emerging fields of reparative and ...regenerative medicine. Currently, a number of media or "solutions" are utilized for the preservation of biologics. Given the diversity of cell systems utilized in the regenerative medicine arena, we hypothesized that the development of unique (individualized) preservation solutions designed to meet the distinct molecular biological requirements of individual systems would provide for enhanced and extended preservation. To evaluate this hypothesis, coronary artery smooth muscle cells (CASMCs), coronary artery endothelial cells (CAECs), hepatic cells (C3A), and skeletal muscle cells (SKMCs) were hypothermically preserved for 2 to 7 days at 4 degrees C in either cell culture medium, University of Wisconsin Solution (UW or ViaSpan), or HypoThermosol (HTS) variants. Cells were then assayed for viability, using the alamarBlue assay as well as calcein-AM, subsequent to their return to normothermic (37 degrees C) temperatures for up to 5 days. CASMC viability was best maintained when preserved in HTS plus Trolox/EDTA, CAEC viability was highest when preserved in HTS plus Trolox, SKMCs stored in HTS plus Trolox/RGD demonstrated enhanced viability, and C3A cells were best preserved in HTS plus FK041. The data suggest that solution compositions that address the differences in cell death mechanisms limiting preservation efficacy can result in targeted improvement matched to specific cell types. These observations support the custom solution hypothesis of cell and tissue preservation.
Adjuvant therapies contribute to the successful treatment of cancer. Our previous reports have shown that combining cryoablation with cytotoxic agents enhances cell death. Tumor necrosis ...factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic agent that preferentially induces apoptosis in a variety of human cancer cells. Human prostate cancer cells (PC-3) are resistant to many cytodestructive agents, including cryoablation and TRAIL. Here, we evaluated the effects of TRAIL combined with cryoablation on PC-3 and normal prostate (RWPE-1) cell death. Exposure of PC-3 cells to freezing (-10 degrees C) or TRAIL (500 ng/ml) results in minimal cell death, whereas a complete loss of viability is observed with the simultaneous combination. The synergistic effect was found to be due to a marked increase in apoptosis. Western blot analysis revealed a significant level of caspase-8 and -3 cleavage between 12 and 24 h post-exposure. Caspase activation assays provided similar results and also indicated a role for caspase-9. Inhibitors to caspase-8 and -9 along with a pan-caspase inhibitor were incorporated to determine which pathway was necessary for the combined efficacy. Inhibition of caspase-8 significantly blocked the combination-induced cell death compared to cells that did not receive the inhibitor (63% compared to 10% viable). The addition of the caspase-9 inhibitor resulted in only a minimal protection. Importantly, the combination was not effective when applied to normal prostate cells. The results describe a novel therapeutic model for the treatment of prostate cancer and provide support for future in vivo studies.
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