In recent study, microRNAs have various important functions in diverse biological processes and progression of cancer. In human breast cancer, microRNA-22 has been reported to be downregulated. However, molecular mechanism of microRNA-22 in breast cancer progression and chemosensitivity has not been well studied. In our study, these results demonstrated that microRNA-22 expression levels were significantly reduced in 40 pairs of human breast cancer tissues when compared to normal tissues. Enforced expression of microRNA-22 inhibited activity of cell proliferation and cell migration in breast cancer cells. Furthermore, microRNA-22 targeted NRAS proto-oncogene, GTPase (NRAS) in breast cancer cells. The expression levels of NRAS in human clinical specimens were higher in breast cancer tissues when compared to normal tissues. Moreover, microRNA-22 sensitized breast cancer cells to paclitaxel by regulation of NRAS. Our results then demonstrated that microRNA-22 functioned as a tumor suppressor microRNA and indicated potential application for the diagnosis and treatment of cancer in the future.
Lung cancer remains one of the most common cancer-related deaths worldwide. The cigarette smoking is a risk factor for lung cancer development. Interestingly, the cystic fibrosis transmembrane conductance regulator encoded by CFTR gene, an ATP-binding cassette transporter-class ion channel that conducts chloride and bicarbonate anions across membrane of epithelial cells, has recently been suggested to play a role in the development and progression of many types of cancer. It has been well-documented that mutations of CFTR gene are the cause of cystic fibrosis, the most common fatal hereditary lung disease in Caucasian population; the function of cystic fibrosis transmembrane conductance regulator in the development of lung cancer however has not yet been established. In the present study, we aimed to interrogate the impact of cystic fibrosis transmembrane conductance regulator on the nicotine-promoted progressive potency in lung adenocarcinoma cells by assessing capacities of cystic fibrosis transmembrane conductance regulator to cell migration, invasion, and clonogenicity and the expression of markers of cell proliferation and lung stem cell-related transcription factors in lung adenocarcinoma A549 cells. The exposure of nicotine exhibited an ability to enhance progressive properties of adenocarcinoma cells including A549 cells, HCC827 cells, and PC-9 cells, alone with an inhibition of cystic fibrosis transmembrane conductance regulator protein expression. Remarkably, an overexpression of cystic fibrosis transmembrane conductance regulator significantly inhibited the progressive potency of A549 cells, including capacity of cell migration and invasion and clonogenicity, along with a decreased expression of cell proliferative markers Ki67, p63, and proliferating cell nuclear antigen, and cancer stem cell marker CD 133, stem cell pluripotency-related transcription factors octamer-binding transcription factor 3/4, and sex-determining region Y-box 2, regardless of the presence of nicotine. In contrast, opposite effects were observed in A549 cells that the cystic fibrosis transmembrane conductance regulator was knockdown by short hairpin RNA to cystic fibrosis transmembrane conductance regulator. This study thus suggests that cystic fibrosis transmembrane conductance regulator may play a tumor suppressor role in lung cancer cells, which may be a novel therapeutic target warranted for further investigation.
HOX transcript antisense intergenic RNA has been reported to serve as an important prognostic biomarker in several types of cancers. However, the clinical value of HOX transcript antisense intergenic RNA in digestive cancers remains unclear. Therefore, we tried to investigate the clinical role of expression of HOX transcript antisense intergenic RNA as a prognostic indicator in digestive cancers by a meta-analysis. Literature collection was performed by searching the PubMed, Embase, Web of Science, and Cochrane Library databases (up to October 7, 2017). A quantitative meta-analysis was conducted to assess the eligible articles on the prognostic value of HOX transcript antisense intergenic RNA in digestive cancers. The pooled hazard ratios or odds ratios with 95% confidence intervals were used to evaluate the association between expression of HOX transcript antisense intergenic RNA and clinical outcomes. A total of 1844 patients from 22 studies were included in this meta-analysis. The results found a significant association between expression of HOX transcript antisense intergenic RNA and poor overall survival in digestive cancers (pooled hazard ratio = 2.19, 95% confidence interval, 1.86-2.57, P < .001). Furthermore, subgroup analysis showed that tumor type, region, Newcastle-Ottawa scale, and sample size did not alter the predictive value of HOX transcript antisense intergenic RNA as an independent factor for patients' survival. In addition, we also revealed that the clinicopathological characteristics such as differentiation, lymph node metastasis, tumor node metastasis (TNM) stage, and distant metastasis were positively related to expression of HOX transcript antisense intergenic RNA digestive cancers. In conclusion, our results suggested high expression of HOX transcript antisense intergenic RNA was correlated with poor clinical outcomes and may serve as a novel prognostic biomarker for patients with digestive cancers.
Osteosarcoma is one of the most common primary malignant bone tumors. The inhibitor of growth family of protein 5 has been identified as a tumor suppressor in many cancers. In this study, we confirmed the downregulation of the both inhibitor of growth family of protein 5 and messenger RNA levels in cancer tissues using Western blot and real-time polymerase chain reaction. In order to find the antitumor roles of inhibitor of growth family of protein 5, osteosarcoma cells, HOS, and MG63 were transfected with the plasmid pCDNA-3.1-inhibitor of growth family of protein 5. Overexpression of Inhibitor of growth family of protein 5 could induce apoptosis and inhibit cell proliferation in osteosarcoma cells. Furthermore, Western blot analysis showed that p-Smad2, p-Smad3, and Smad4 were increased in inhibitor of growth family of protein 5-expressing osteosarcoma cells. Our results indicated that overexpression of inhibitor of growth family of protein 5 in osteosarcoma cells induces apoptosis by activating the Smad pathway, thus proposing a promising role for inhibitor of growth family of protein 5 in treatment of patients with osteosarcoma.
Mutations in the AT-rich interactive domain 1A gene, which encodes a subunit of the Switch/Sucrose nonfermentable chromatin remodeling complex, can result in loss of protein expression and are associated with different cancers. Here, we used immunohistochemistry to investigate the significance of AT-rich interactive domain 1A loss in 73 pancreatic ductal adenocarcinoma cases with paired paracancerous normal pancreatic tissues. The relationship between levels of the AT-rich interactive domain 1A protein product, BAF250a, and clinicopathological parameters in the 73 pancreatic cancer specimens was also analyzed. We found that the expression of AT-rich interactive domain 1A in normal pancreatic tissue was higher than that in tumor tissue. Loss of AT-rich interactive domain 1A expression in pancreatic tumors was associated with tumor differentiation (P = .002) and tumor stage (P = .048). Meanwhile, BAF250a protein levels were not related to lymph node metastasis, distant metastasis, sex, or age and were not associated with survival. Transfection of the pancreatic cancer cell lines AsPC-1 and PANC-1 with small-interfering RNA specific for AT-rich interactive domain 1A resulted in elevated messenger RNA and protein expression levels of B-cell lymphoma-2 (Bcl-2), CyclinD1, and Kirsten rat sarcoma viral oncogene (KRAS). The AT-rich interactive domain 1A expression level in the cells was increased following microRNA-31 (miR-31) inhibitor transfection. Our data provide additional evidence that AT-rich interactive domain 1A might function as a tumor suppressor gene in pancreatic carcinogenesis.
The abnormal phosphatase and tensin homolog expression and activated phosphoinositide-3 kinase/Protein kinase B (AKT)/mammalian target of rapamycin signaling pathway are involved in the progression of esophageal squamous cell carcinoma. By assessing the expression pattern of key components in the phosphoinositide-3 kinase/AKT/mammalian target of rapamycin signaling pathway by immunohistochemistry in tumor and nontumor esophageal mucosa from patients with esophageal squamous cell carcinomas, we aimed to carefully explore the relationship between the various protein expressions and clinicopathological factors, as well as patient outcome. A total of 145 tumor and 145 nontumor samples from patients with esophageal squamous cell carcinoma, collected from HuaShan Hospital (Shanghai, China) were evaluated. Clinical characteristics, the targeted protein expressions (including phosphatase and tensin homolog, phosphoinositide-3 kinase, AKT, p-AKT, mammalian target of rapamycin, p-mTOR, p70S6 kinase 1, p-P70S6K1, elongation initiation factor 4E binding protein-1, and p-4E-BP1, and survival rate were analyzed. Among them, phosphoinositide-3 kinase, AKT, p-AKT, mammalian target of rapamycin, p-mTOR, elongation initiation factor 4E binding protein-1, p70S6 kinase 1, and p-P70S6K1 proteins were significantly upregulated in tumor tissue. Conversely, phosphatase and tensin homolog was largely downregulated in tumor tissue, notably in pT3-T4 tumors. Low expression of phosphatase and tensin homolog whereas high expression of mammalian target of rapamycin signaling components in tumors was closely related to the presence of lymph node metastases and advanced TNM stage (all P < .05). Moreover phosphatase and tensin homolog, mammalian target of rapamycin, and p70S6 kinase 1 were correlated with overall survival as well as p-mTOR was correlated with progression-free survival (all P < .05). Overexpression of mammalian target of rapamycin was proved to be an independent adverse prognostic factor for overall survival in esophageal squamous cell carcinomas. Our results suggest that the phosphoinositide-3 kinase/AKT/mammalian target of rapamycin signaling pathway is activated in esophageal squamous cell carcinoma, with the low expression of phosphatase and tensin homolog and the high expression of the mammalian target of rapamycin component proteins (both total and phosphorylated) in tumor tissue. Our result might offer a new strategy for specific targeted therapy and prognostic assessment in esophageal cancer.
The pathogenesis of hepatocellular carcinoma is complex and not fully known yet. This study aims to screen and identify the differentially expressed proteins in peripheral blood and liver tissue samples from rat hepatocellular carcinoma and to further clarify the pathogenesis and discover the specific tumor markers and molecular targets of hepatocellular carcinoma. The hepatocellular carcinoma model of Wistar rats were induced by chemical carcinogen. The serum and liver tissue samples were obtained after induction for 2, 4, 8, 14, 18, and 21 weeks. The results showed that the clusterin (IPI00198667), heat shock protein a8 (IPI00208205), and N-myc downstream-regulated gene-2 (IPI00382069) being closely related to hepatocarcinogenesis were eventually identified from the 30 different proteins. As the time progressed, the serum levels of clusterin and heat shock protein a8 increased gradually during induced liver cancer in rats. However, the serum N-myc downstream-regulated gene 2 level in induced liver cancer in rats underwent biphasic changes, and the serum N-myc downstream-regulated gene 2 level decreased at the 8th week, increased at the 14th week, and then decreased significantly. Statistical difference occurred in protein expression of clusterin and heat shock protein a8 in liver tissues at the different time points. In the liver tissues, the N-myc downstream-regulated gene 2 level decreased gradually at the 8th week, increased gradually at the 14th week, and then decreased significantly after 14 weeks. The study demonstrated that heat shock protein a8, clusterin, and N-myc downstream-regulated gene 2 participated in the process of abnormal cell division, proliferation, and carcinogenesis of liver cells during hepatocarcinogenesis.
Objective: This study aims to investigate the correlations between rapidly accelerated fibrosarcoma/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase signaling pathway and clinicopathological features and prognosis for patients with breast cancer having axillary lymph node metastasis. Methods: A total of 118 breast cancer tissues with axillary lymph node metastasis (axillary lymph node metastasis group), 150 breast cancer tissues with non-axillary lymph node metastasis (non-axillary lymph node metastasis group), and 216 normal breast tissues (normal group) were enrolled in this study. The messenger RNA and protein expressions of rapidly accelerated fibrosarcoma, MEK, extracellular signal-regulated kinase, and their phosphorylated (p-) proteins were examined by reverse transcriptase quantitative polymerase chain reaction and immunohistochemistry, respectively. All patients received a 1-year follow-up, and the clinical follow-up data were collected. The multiple factors on the prognosis of patients with breast cancer having axillary lymph node metastasis were tested by Cox regression analysis. Results: The messenger RNA expressions of rapidly accelerated fibrosarcoma, MEK, and extracellular signal-regulated kinase and positive rates of rapidly accelerated fibrosarcoma, MEK, phosphorylated MEK, extracellular signal-regulated kinase, and p-extracellular signal-regulated kinase in the axillary lymph node metastasis group were higher than in the non-axillary lymph node metastasis and normal groups (all P < .05). The protein expressions of rapidly accelerated fibrosarcoma, MEK, phosphorylated MEK, extracellular signal-regulated kinase, and p-extracellular signal-regulated kinase were associated with tumor size, clinical stage, and axillary lymph node metastasis number (all P < .05). Rapidly accelerated fibrosarcoma, MEK, and extracellular signal-regulated kinase expressions were significantly correlated with the prognosis of patients with breast cancer (all P < .05). Patients with BC having positive rapidly accelerated fibrosarcoma, MEK, phosphorylated MEK, extracellular signal-regulated kinase, and phosphorylated ERK expressions had a higher survival rate than patients with BC having the negative ones (all P < .05). Rapidly accelerated fibrosarcoma and extracellular signal-regulated kinase protein expressions, clinical stage, pathological grade, and axillary lymph node metastasis number were independent prognostic factors in patients with breast cancer having axillary lymph node metastasis (all P < .05). Conclusion: Our study proved that rapidly accelerated fibrosarcoma/MEK/extracellular signal-regulated kinase signaling pathway is significantly correlated with the clinicopathological features and prognosis for patients with BC having axillary lymph node metastasis. Rapidly accelerated fibrosarcoma and extracellular signal-regulated kinase protein expressions are independent prognostic factors for patients with breast cancer having axillary lymph node metastasis.
Purpose: We performed this network meta-analysis to compare the diagnostic value of 4 imaging methods (magnetic resonance imaging, positron emission tomography, computed tomography, and diffusion-weighted imaging) for diagnosing lymph node metastases in cervical cancer. Method: Diagnostic tests regarding different imaging methods to diagnose lymph node metastases in cervical cancer were retrieved from the Cochrane Library, PubMed, and Embase electronic databases from inception to December 2016. Direct and indirect evidence was performed to calculate the odds ratio and to draw the surface under the cumulative ranking curves of the 4 imaging methods for diagnosing lymph node metastases in cervical cancer. Results: Sixteen eligible diagnostic tests were included in this network meta-analysis. The results of network meta-analysis demonstrate that in comparison with the diffusion-weighted imaging, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio of positron emission tomography were relatively higher. Additionally, the results further indicate that compared with other diagnosis method, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio of positron emission tomography had a higher trend. The surface under the cumulative ranking curve results indicated that in terms of positive likelihood ratio and diagnostic odds ratio, positron emission tomography had a relatively higher diagnostic value for lymph node metastases in patients with cervical cancer. Conclusion: Our findings indicate that positron emission tomography might have a relatively higher diagnostic value for lymph node metastases in patients with cervical cancer.
Objective: The aim of this study was to evaluate the mechanisms involved with miRNA-708 and its targeting of bone morphogenetic protein and activin membrane-bound inhibitor in cell proliferation, migration, and apoptosis in mice with melanoma via the Wnt and transforming growth factor beta signaling pathways. Methods: Sixty mice were recruited of which 40 were subsequently assigned into the experimental group (22 mice were successfully established as melanoma model and 18 mice used in tumor xenograft), and the normal control group consisted of 20 mice. B16 cells were assigned to the normal, blank, and negative control, miR-708 mimics, miR-708 inhibitors, si-BAMBI, and miR-708 inhibitors thorn si-bone morphogenetic protein and activin membrane-bound inhibitor groups. Western blotting and reverse transcription quantitative polymerase chain reaction were employed to detect the expression levels within the tissues and cell lines. TCF luciferase reporter (TOP-FLASH) or a control vector (FOP-FLASH) was applied to detect the activity of the Wnt signaling pathway. MTT3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay, flow cytometry, scratch test, and Transwell assay were conducted, respectively, for cell proliferation, apoptosis, migration, and invasion, while tumor xenograft procedures were performed on the nude mice recruited for the study. Results: Compared to the normal control group, the model group displayed increased expressions of bone morphogenetic protein and activin membrane-bound inhibitor, Wnt10B, P53, and Bcl-2; TOPflash activity; beta-catenin expression; cell proliferation; migration; and invasion capabilities while decreased expressions of miR-708, vascular endothelial growth factor, Fas, Bax, Caspase-3, and cleaved Caspase-3 and apoptosis rate. Compared to the blank and negative control groups, the miR-708 mimics and small-interfering RNA-bone morphogenetic protein and activin membrane-bound inhibitor groups exhibited decreases expressions of bone morphogenetic protein and activin membrane-bound inhibitor, Wnt10B, P53, and Bcl-2 and decreased proliferation, migration, and invasion capabilities, while increases in the apoptosis rate, expressions of vascular endothelial growth factor, Fas, Bax, Caspase-3, and cleaved Caspase-3; however, downregulated levels of TOPflash activity and beta-catenin expression were recorded. The miR-708 inhibitors group displayed an opposite trend. Conclusion: Downregulation of miR-708-targeted bone morphogenetic protein and activin membrane-bound inhibitor inhibits the proliferation and migration of melanoma cells through the activation of the transforming growth factor beta pathway and the suppression of Wnt pathway.
Staphylococcal enterotoxin A is well known as a superantigen and able to be used for cancer immunotherapy. In this study, recombinant Staphylococcal enterotoxin A was genetically conjugated to epidermal growth factor to produce a chimeric protein recombinant Staphylococcal enterotoxin A-epidermal growth factor expressed in Escherichia coli. The recombinant Staphylococcal enterotoxin A-epidermal growth factor protein was purified using Strep-Tactin affinity chromatography and Endotoxin Removal Resin and identified by sodium dodecyl sulfate-polyacrylamide gel electropheresis and liquid chromatography-tandem mass spectrometry analysis. Furthermore, in vitro experiments showed purified recombinant Staphylococcal enterotoxin A-epidermal growth factor could successfully bind to the human nasopharyngeal carcinoma cell line CNE2, significantly promote the proliferation of human peripheral blood mononuclear cells, and enhance the secretion of several cytokines that have broad antitumor activities, such as interferon-gamma, tumor necrosis factor-alpha, and interleukin-2. Importantly, recombinant Staphylococcal enterotoxin A-epidermal growth factor significantly inhibited proliferation of CNE2 cells and promoted apoptosis in CNE2 cells when cocultured with peripheral blood mononuclear cells. Finally, both the binding of recombinant Staphylococcal enterotoxin A-epidermal growth factor and the toxicity of recombinant Staphylococcal enterotoxin A-epidermal growth factor-activated peripheral blood mononuclear cells were demonstrated as specific and only effective on high epidermal growth factor receptor-expressing cell lines. In all, our work suggests that recombinant Staphylococcal enterotoxin A-epidermal growth factor serves as a promising novel immunotherapeutic agent. More in vivo and in vitro studies are needed to verify its antitumor potency, as well as investigate the underlying mechanisms in cancer immunotherapy.
MicroRNAs have been reported to play an important role in diverse biological processes and progression of various cancers. MicroRNA-29a has been observed to be downregulated in human lung cancer tissues, but the function of microRNA-29a in lung cancer has not been well investigated. In this study, we demonstrated that the expression levels of microRNA-29a were significantly downregulated in 38 pairs of lung cancer tissues when compared to adjacent normal tissues. Overexpression of microRNA-29a inhibited the activity of cell proliferation and colony formation of lung cancer cells, H1299 and A549. Furthermore, microRNA-29a targeted NRAS proto-oncogene in lung cancer cells. In human clinical specimens, NRAS proto-oncogene was highly expressed in human lung cancer tissues compared to normal tissues. More interestingly, microRNA-29a also sensitizes lung cancer cells to cisplatin (CDDP[Please replace "CDDP" with its expansion in the abstract and also provide expansion for the same in its first occurrence in text, if appropriate.]) via its target, NRAS proto-oncogene. Thus, our results in this study demonstrated that microRNA-29a acted as a tumor suppressor microRNA, which indicated potential application of microRNAs for the treatment of human lung cancer in the future.
The relationship between vascular endothelial growth factor gene polymorphism and gastric cancer risk and its development, prognosis, and survival are still being debated. This meta-analysis was performed to assess these relationships. The association reports were identified from PubMed, Embase, Cochrane Library, and CBM-disc (China Biological Medicine Database), and eligible studies were included and calculated using the meta-analysis method. VEGF+936C/T, VEGF+405 G>C, VEGF-460 T>C, VEGF-1498 T>C, and VEGF-2578 C>A gene polymorphisms were found to be unassociated with gastric cancer risk for the overall population in this meta-analysis, whereas the VEGF-634 G>C GG genotype was associated with gastric cancer risk in the overall population. Furthermore, VEGF-634 G>C C allele and the GG genotype were associated with gastric cancer risk in Caucasians, and VEGF+1612G/A gene polymorphism was associated with gastric cancer risk for the Asian population. VEGF+936C/T gene polymorphism was not associated with the stage of cancer, lymph node metastasis, Lauren classification, or survival of gastric cancer. However, VEGF+936C/T T allele and TT genotype were associated with the tumor size of gastric cancer. In conclusion, the VEGF-634 G>C GG genotype was associated with gastric cancer risk in the overall population with the VEGF-634 G>C C allele and GG genotype being associated with risk in Caucasians and VEGF+1612G/A in the Asian population.
Radiotherapy is the primary treatment for nasopharyngeal carcinoma while radioresistance can hinder efficient treatment. To explore the role of annexin A1 and its potential mechanisms in radioresistance of nasopharyngeal carcinoma, human nasopharyngeal carcinoma cell line CNE2-sh annexin A1 (knockdown of annexin A1) and the control cell line CNE2-pLKO. 1 were constituted and CNE2-sh annexin A1 xenograft mouse model was generated. The effect of annexin A1 knockdown on the growth of xenograft tumor after irradiation and radiation-induced DNA damage and repair was analyzed. The results of immunohistochemistry assays and Western blotting showed that the level of annexin A1 was significantly downregulated in the radioresistant nasopharyngeal carcinoma tissues or cell line compared to the radiosensitive nasopharyngeal carcinoma tissues or cell line. Knockdown of annexin A1 significantly promoted CNE2-sh annexin A1 xenograft tumor growth compared to the control groups after irradiation. Moreover, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays revealed that knockdown of annexin A1 significantly inhibited apoptosis in vivo compared to the control groups. We assessed the intracellular reactive oxygen species levels and the extent of radiation-induced DNA damage and repair using reactive oxygen species assay, comet assays, and immunohistochemistry assay. The results showed that knockdown of annexin A1 remarkedly reduced the intracellular reactive oxygen species levels, level of DNA double-strand breaks, and the phosphorylation level of H2AX and increased the accumulation of DNA-dependent protein kinase in nasopharyngeal carcinoma cells after irradiation. The findings suggest that knockdown of annexin A1 inhibits DNA damage via decreasing the generation of intracellular reactive oxygen species and the formation of g-H2AX and promotes DNA repair via increasing DNA-dependent protein kinase activity and therefore improves the radioresistance in nasopharyngeal carcinoma cells. Together, our findings suggest that knockdown of annexin A1 promotes radioresistance in nasopharyngeal carcinoma and provides insights into therapeutic targets for nasopharyngeal carcinoma radiotherapy.
The aim of the study was to perform cryosurgery on a primary breast tumor, coupled with simultaneous peritumoral and intratumoral tracer injection of a blue dye, to evaluate lymphatic mapping. We explored the ability of our strategy to prevent tumor cells, but not that of injected tracers, to migrate to the lymphovascular drainage during conventional resection of frozen breast malignancies. Seventeen patients aged 51 (14) years (mean [standard deviation]), presenting primary breast cancer with stage I to IV, were randomly selected and treated in The Rudolfinerhaus Private Clinic in Vienna, Austria, and included in this preliminary clinical study. Under intraoperative ultrasound, 14 patients underwent curative cryo-assisted tumor resection en bloc, coupled with peritumoral tracer injection, which consisted of complete tumor freezing and concomitant peritumor injection with a blue dye, before resection and sentinel lymph node dissection (group A). Group B consists of 3 patients previously refused any standard therapy and had palliative tumor cryoablation in situ combined with intratumoral tracer injection. The intraoperative ultrasound facilitated needle positioning and dye injection timing. In group A, the frozen site extruded the dye that was distributed through the unfrozen tumor, the breast tissue, and the resection cavity for 12 patients. One to 4 lymph nodes were stained for 10 of 14 patients. The resection margin was evaluable. Our intraoperative ultrasound-guided performance revealed the injection and migration of a blue dye during the frozen resection en bloc and cryoablation in situ of primary breast tumors. Sentinel lymph node mapping, pathological determination of the tumor, and resection margins were achievable. The study paves the way for intraoperative cryo-assisted therapeutic strategies for breast cancer.