The purpose of this study was to compare the in vivo anti-tumor efficacy of a mucoadhesive, lipid-based, oral paclitaxel formulation (DHP107) with traditional, intraperitoneal (IP) paclitaxel using an orthotopic mouse model of chemotherapy-sensitive SKOV3ip1 ovarian cancer.
To determine the optimal therapeutic dose of oral paclitaxel, DHP107 was administered per os to female athymic nude mice at 0, 25, or 50 mg/kg twice per week. Control mice received 100 µL saline once per week. IP injections of paclitaxel at 5 mg/kg once per week were used for comparison. To evaluate the potential therapeutic effect of metronomic DHP107 chemotherapy, mice received DHP107 50 mg/kg once per week per os, which was compared with 25 mg/kg twice per week and with vehicle-treated controls.
Low-dose DHP107 (25 mg/kg) twice per week was as effective as IP paclitaxel (5 mg/kg once a week) but high-dose DHP107 (50 mg/kg once per week) was less effective at inhibiting tumor growth in an orthotopic mouse model (88%, 82%, and 36% decrease in tumor weight, respectively). Mice that received 25 mg/kg DHP107 twice per week or 50 mg/kg DHP107 once per week per os had a significant decrease in tumor weight compared with vehicle-treated controls (p<0.01, both doses).
Metronomic oral chemotherapy with DHP107 showed anti-tumor efficacy in vivo similar to IP paclitaxel in an orthotopic mouse model.
Chemotherapy; DHP107; Oral paclitaxel; Ovarian cancer
Changes of glycosylation pattern in serum proteins have been linked to various diseases including cancer, suggesting possible development of novel biomarkers based on the glycomic analysis. In this study, N-linked glycans from human serum were quantitatively profiled by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and compared between healthy controls and ovarian cancer patients. A training set consisting of 40 healthy controls and 40 ovarian cancer cases demonstrated an inverse correlation between P value of ANOVA and area under the curve (AUC) of each candidate biomarker peak from MALDI-TOF MS, providing standards for the classification. A multibiomarker panel composed of 15 MALDI-TOF MS peaks resulted in AUC of 0.89, 80~90% sensitivity, and 70~83% specificity in the training set. The performance of the biomarker panel was validated in a separate blind test set composed of 23 healthy controls and 37 ovarian cancer patients, leading to 81~84% sensitivity and 83% specificity with cut-off values determined by the training set. Sensitivity of CA-125, the most widely used ovarian cancer marker, was 74% in the training set and 78% in the test set, respectively. These results indicate that MALDI-TOF MS-mediated serum N-glycan analysis could provide critical information for the screening of ovarian cancer.
The purpose of this study was to investigate whether selective cyclooxygenase (COX) inhibitors promote paclitaxel-induced apoptosis in taxane-resistant ovarian cancer cells by suppressing MDR1/P-glycoprotein (P-gp) expression.
Taxane-resistant ovarian cancer cells were cultured with paclitaxel alone or combined with a selective COX inhibitors. The expression patterns of MDR1/P-gp and the ability of COX inhibitors to inhibit growth of taxane-resistant ovarian cancer cells were measured. The efficacy of prostaglandin E2 (PGE2) supplementation was measured to evaluate the mechanisms involved in suppressing MDR1 gene expression.
P-gp was upregulated in taxane-resistant ovarian cancer cells compared to paired paclitaxel-sensitive ovarian cancer cells. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that selective COX inhibitors significantly enhanced the cytotoxic effects of paclitaxel in taxane-resistant ovarian cancer cells via a prostaglandin-independent mechanism. These increased apoptotic effects were further verified by measuring an increased percentage of cells in sub-G1 stage using flow cytometry. Selective COX inhibitors suppressed MDR1 and P-gp expression. Moreover, combined treatment with paclitaxel and selective COX inhibitors increased poly (ADP-ribose) polymerase (PARP) cleavage in taxane-resistant ovarian cancer cells.
Selective COX inhibitors significantly promote paclitaxel-induced cell death in taxane-resistant ovarian cancer cells in a prostaglandin-independent manner. COX inhibitors could be potent therapeutic tools to promote paclitaxel sensitization of taxane-resistant ovarian cancers by suppressing MDR1/P-gp, which is responsible for the efflux of chemotherapeutic agents.
Chemosensitizer; Cyclooxygenase inhibitor; Ovarian cancer; Paclitaxel; P-glycoprotein
B-1 cells, which constitute a predominant lymphocyte subset in serosal cavities and produce most of natural antibodies, are subdivided into the CD5+ B-1a and CD5- B-1b cell subpopulations, but the differential roles of B-1a and B-1b cells are not well understood. We report that B-1a cells preferentially migrate out of the peritoneal cavity and upregulate the expression of CXCR4 with heightened sensitivity to CXCL12 and CXCL13 upon LPS treatment compared to B-1b and B-2 cells. Whereas B-1a cells were slightly more abundant than B-1b and B-2 cells in the homeostatic condition, the number of B-1a cells preferentially decreased 48 hr after LPS treatment. The decrease in the peritoneal B-1a cell number was accompanied with increased migration of B-1a cells toward CXCL-12 and CXCL-13 in in vitro transmigration assay using peritoneal B cells from LPS treated mice. The expression level of CXCR4, but not of CXCR5, was also more prominently increased in B-1a cells upon LPS stimulation. LPS-stimulated B-1a cells did not accumulate in omental milky spots in contrast to B-2 cells. These results suggest that B-1a cells actively migrate out of the peritoneal cavity through the regulation of the migratory responsiveness to chemokines and actively participate in systemic immune responses.
B Lymphocyte Subsets; Chemokine CXCL12; Chemokine CXCL13; Chemotaxis; Receptor, CXCR4; Lipopolysaccharides
To evaluate the clinicopathologic characteristics and prognostic factors of ovarian granulosa cell tumors.
Medical records of 113 patients presenting between January 1995 and December 2007 were retrospectively reviewed.
One-hundred two patients had adult type disease, with a mean age of 46.2 years (range, 18 to 83 years) and a mean follow-up period of 54.7 months (range, 1 to 155 months). The distribution of FIGO stages was 86 patients at stage I, 11 at stage II, and 5 at stage III. During follow-up, ten patients recurred at a mean time of 48 months (range, 4 to 109 months). Among them, three patients died after a mean of 57 months (range, 25 to 103 months). In recurrence analysis, advanced stage (p=0.032) and presence of residual disease (p=0.012) were statistically significant, and age<40 years, premenopause and positive washing cytology were marginally significant (p<0.1). In multivariate analysis, stage was the only factor associated with recurrence; adjuvant chemotherapy and fertility-sparing surgery were not statistically significant. Among 36 patients with fertility-sparing operations, eight patients had nine pregnancies and delivered seven babies. Eleven patients had juvenile type tumors; the mean age was 20.0 years (range, 8 to 45 years) and the mean follow-up period was 69.8 months (range, 20 to 156 months). The distribution of FIGO stage was nine patients at stage I and two at stage III. There were no recurrences or deaths reported. Four patients had seven pregnancies and delivered six babies.
Stage is the only factor associated with disease-free survival, and fertility-sparing surgery may be a treatment option for women with early-stage disease who want to retain fertility.
Clinical study; Granulosa cell tumor; Ovary; Pregnancy; Recurrence
To evaluate the occurrence of residual or recurrent disease after conization for adenocarcinoma in situ (AIS) of the uterine cervix.
Medical records of 99 patients with a histologically diagnosis of AIS of the uterine cervix by conization between 1991 and 2008 were reviewed retrospectively.
Seventy eight of 99 patients (78.8%) had negative and 18 (18.2%) had positive resection margins of the conization specimen, and 3 (3.0%) had unknown margin status. Of the 78 patients with negative margins, 45 underwent subsequent hysterectomy and residual AIS were present in 4.4% (2/45) of patients. Ten of the 18 patients with positive margins received subsequent hysterectomy and 3 patients (30%) had residual AIS. Twenty-eight patients had conservative treatment and during the median follow-up time of 23.5 months (range, 7 to 124 months), only one patient (3.6%) had recurrent AIS and was treated with a simple hysterectomy. Eight patients became pregnant after conization, 4 of them delivered healthy babies, one had a spontaneous abortion and 3 were ongoing pregnancies.
Patients with positive resection margins after conization for AIS of the uterine cervix are significantly more likely to have residual disease. However, negative resection margin carries a lower risk for residual AIS, therefore conservative management with careful surveillance seems to be feasible in women who wish to preserve their fertility.
Adenocarcinoma in situ; Uterine cervix cancer; Conization; Conservative treatment
Lymphotoxin expressed by RORγt+ innate lymphoid cells is critical for natural killer cell development.
Natural killer (NK) cell development relies on signals provided from the bone marrow (BM) microenvironment. It is thought that lymphotoxin (LT) α1β2 expressed by the NK cell lineage interacts with BM stromal cells to promote NK cell development. However, we now report that a small number of RORγt+ innate lymphoid cells (ILCs), and not CD3−NK1.1+ cells, express LT to drive NK development. Similar to LT−/− or RORγt−/− mice, the mice conditionally lacking LTα1β2 on RORγt+ ILCs experience a developmental arrest at the immature NK stages, between stages of NK development to the mature NK cell stage. This developmental block results in a functional deficiency in the clearance of NK-sensitive tumor cells. Reconstitution of Thy1+ ILCs from BM or purified RORγt+ ILCs from lamina propria lymphocytes into LT-deficient RORγt+ BM cultures rescues NK cell development. These data highlight a previously undiscovered role of RORγt+ ILCs for NK cell development and define LT from ILCs as an essential molecule for the stromal microenvironment supporting NK cell development.
While stationary organ cells are in continuous contact with neighboring cells, immune cells circulate throughout the body without an apparent requirement for cell-cell contact to persist in vivo. This study challenges current convention by demonstrating, both in vitro and in vivo, that innate immune NK cells can engage in homotypic NK-to-NK cell interactions for optimal survival, activation, and proliferation. Using a specialized cell-laden microwell approach, we discover that NK cells experiencing constant NK-to-NK contact exhibit a synergistic increase in activation status, cell proliferation, and anti-tumor function in response to IL-2 or IL-15. This effect is dependent on 2B4/CD48 ligation and an active cytoskeleton, resulting in amplification of IL-2 receptor signaling, enhanced CD122/CD132 colocalization, CD25 upregulation, and Stat3 activation. Conversely, ‘orphan' NK cells demonstrate no such synergy and fail to persist. Therefore, our data uncover the existence of homotypic cell-to-cell communication among mobile innate lymphocytes, which promotes functional synergy within the cytokine-rich microenvironment.
Here, we demonstrate that electroporation-enhanced immunization with a rationally designed HPV DNA vaccine (GX-188E), preferentially targeting HPV antigens to dendritic cells, elicits a significant E6/E7-specific IFN-γ-producing T-cell response in all nine cervical intraepithelial neoplasia 3 (CIN3) patients. Importantly, eight out of nine patients exhibit an enhanced polyfunctional HPV-specific CD8 T-cell response as shown by an increase in cytolytic activity, proliferative capacity and secretion of effector molecules. Notably, seven out of nine patients display complete regression of their lesions and viral clearance within 36 weeks of follow up. GX-188E administration does not elicit serious vaccine-associated adverse events at all administered doses. These findings indicate that the magnitude of systemic polyfunctional CD8 T-cell response is the main contributing factor for histological, cytological and virological responses, providing valuable insights into the design of therapeutic vaccines for effectively treating persistent infections and cancers in humans.
While several human papilloma virus (HPV) vaccines exist, a highly effective vaccine that mediates regression of HPV-induced tumours is lacking. Here the authors show that a therapeutic DNA vaccine-induced HPV-specific polyfunctional CD8 T cell in 7 out of 9 patients who all exhibited complete regression of lesions and viral clearance.
To assess the effect of single-dose cisplatin intraperitoneally administered during cytoreductive surgery in advanced epithelial ovarian cancer.
Data from patients who underwent surgical management followed by intravenous (IV) chemotherapy for stage III epithelial ovarian cancer from 2003 to 2012 were retrospectively reviewed. Subjects were divided into intraperitoneal (IP) and no-intraperitoneal (NIP) groups according to the administration of IP cisplatin 100 mg during the staging surgery. Clinical results such as survival outcomes and chemotherapeutic toxicity were compared between the two groups.
Thirty-seven patients in the IP group and 26 in the NIP group were identified. There were no significant differences between the two groups in basic characteristics such as age, histology, and surgical procedures. After the surgery with or without IP chemotherapy, there was no difference in the rate of either hematologic or gastrointestinal toxicity or in the rate of incompletion of following IV chemotherapy. Tumor recurrence occurred in 67.6% (25 patients) of IP group and 57.7% (15 patients) of NIP group (P=0.423) during the mean follow-up period of 37 months. The 3-year disease free-survival rate was 39.9% in the IP group and 35.8% in the NIP group, and the relative risk of recurrence was 0.864 (95% confidence interval, 0.447-1.673; P=0.665) in the IP group as compared with the NIP group.
IP chemotherapy with single-dose cisplatin during cytoreductive surgery is safe and feasible with little chemotherapeutic toxicity in advanced epithelial ovarian cancer, but no distinct improvement in survival could be demonstrated in the present study.
Intraperitoneal chemotherapy; Ovarian neoplasms; Survival outcome; Toxicity
Mesenchymal stem cells (MSCs) are a promising candidate for cell-based therapy in regenerative medicine. These stem cells can interact with their mechanical microenvironment to control their functions. External mechanical cues can be perceived and transmitted into intracellular calcium dynamics to regulate various cellular processes. Recent studies indicate that human MSCs (hMSCs) exhibit a heterogeneous nature with a subset of hMSCs lacking spontaneous calcium oscillations. In this study, we studied whether and how external mechanical tension can be applied to trigger and restore the intracellular calcium oscillation in these hMSCs lacking spontaneous activities. Utilizing the fluorescence resonance energy transfer (FRET) based calcium biosensor, we found that this subpopulation of hMSCs can respond to a prolonged mechanical stretch (PMS). Further results revealed that the triggering of calcium oscillations in these cells is dependent on the calcium influx across the plasma membrane, as well as on both cytoskeletal supports, myosin light chain kinase (MLCK)-driven actomyosin contractility, and phospholipase C (PLC) activity. Thus, our report confirmed that mechanical tension can govern the intracellular calcium oscillation in hMSCs, possibly via the control of the calcium permeability of channels at the plasma membrane. Our results also provide novel mechanistic insights into how hMSCs sense mechanical environment to regulate cellular functions.
We analyzed whether expansion of existing active surveillance (AS) protocols to include men with biopsy Gleason score (GS) 3+4 prostate cancer (PCa) would significantly alter pathologic and biochemical outcomes of potential candidates of AS.
Among patients who underwent radical prostatectomy at our center between 2006 and 2013, we identified 577 patients (group A) who preoperatively fulfilled at least one of 6 different AS criteria. Also, we identified 217 patients (group B) with biopsy GS 3+4 but fulfilled non-GS criteria from at least one of 6 AS criteria. Designating group C as expanded group incorporating all patients in group A and B, we compared risk of unfavorable disease (pathologic GS ≥4+3 and/or pathologic T stage ≥pT3a) and biochemical recurrence (BCR)-free survival between groups.
Rates of unfavorable disease were not significantly different between patients of group A and C who met AS criteria from 5 institutions (all p>0.05), not including University of Toronto (p<0.001). Also BCR-free survivals were not significantly different between patients in group A and C meeting each of 6 AS criteria (all p>0.05). Among group B, PSAD>0.15 ng/mL/cm3 (p = 0.011) and tumor length of biopsy GS 3+4 core>4 mm (p = 0.007) were significant predictors of unfavorable disease. When these two criteria were newly applied in defining group B, rates of unfavorable disease in group A and B was 15.6% and 14.7%, respectively (p = 0.886).
Overall rate of pathologically aggressive PCa harbored by potential candidates for AS may not be increased significantly with expansion of criteria to biopsy GS 3+4 under most contemporary AS protocols. PSAD and tumor length of biopsy GS 3+4 core may be useful predictors of more aggressive disease among potential candidates for AS with biopsy GS 3+4.
We sought to investigate the clinicopathologic features of ovarian squamous cell carcinomas arising from mature cystic teratomas (MCT) and to report our clinical experience and lessons learned.
From January 1993 to November 2012, a total of 6,260 women with ovarian MCT were surgically treated at Cheil General Hospital and Women's Healthcare Center. Among them, the cases with malignant transformation to squamous cell carcinoma were included in this analysis. Patient demographic characteristics, surgical findings, and prognosis were evaluated retrospectively.
Of the 6,260 ovarian MCT patients, four (0.06%) had ovarian squamous cell carcinoma arising from MCT. The mean patient age was 43 years (range, 35-51 years), and the mean tumor size was 12 cm (range, 9-16 cm), with two patients in the International Federation of Gynecology and Obstetrics stage I and the other two in stage III. Upon preoperative imaging, all cases were expected to be benign ovarian tumors, but the preoperative squamous cell carcinoma antigen level was elevated from 1.5 ng/mL in stage Ia to 11.3 ng/mL in stage IIIc, suggesting malignancy, while the CA-125 level was normal in two of the three patients who received the test. Optimal debulking surgery was performed and adjuvant chemotherapy was used in all patients, but death from the recurrence of disease occurred in one patient, whose overall survival was 10 months.
Ovarian squamous cell carcinoma arising from MCT is extremely rare, and it is rarely diagnosed preoperatively on imaging workups. Measuring the squamous cell carcinoma antigen level might be a useful diagnostic clue, and it might also be predictive of the tumor stage. An adequate staging surgery should be included in the standard treatment, but multicenter studies are needed to confirm this.
Malignant transformation; Mature cystic teratoma; Ovary; Squamous cell carcinoma
To investigate the surgical and oncological outcomes of laparoscopic surgery compared with laparotomy for the treatment of early-stage ovarian cancer.
Data from patients who underwent surgical management for early-stage ovarian cancer between 2006 and 2012 were retrospectively reviewed. All patients presented with stage I or II disease, and underwent comprehensive staging surgery consisting of a total hysterectomy, bilateral salpingo-oophorectomy, pelvic and para-aortic lymphadenectomy, omentectomy, and peritoneal cytology.
Seventy-seven patients who underwent laparoscopic surgery (24 patients) or laparotomy (53 patients) were identified. Surgery for none of the patients was converted from laparoscopy to laparotomy. The mean operation time was shorter and the estimated blood loss was lower in the laparoscopy group than in the laparotomy group, though the differences were not statistically significant (193 min vs. 224 min, p=0.127; 698 mL vs. 973 mL, p=0.127). There were no differences in the intraoperative or postoperative complications. During a mean follow-up period of 31 months, tumor recurrence occurred in 4 patients: 2 (8.3%) in the laparoscopy group and 2 (3.8%) in the laparotomy group. The mean disease-free survival was 59 months after laparoscopy and 66 months after laparotomy (p=0.367).
Laparoscopic surgery seems to be adequate and feasible for the treatment of early-stage ovarian cancer with comparable results to laparotomy in terms of the surgical outcomes and oncological safety.
Early-stage ovarian cancer; Laparoscopy surgery; Laparotomy staging
Cardiac papillary fibroelastomas (CPF) are benign cardiac tumors and usually discovered incidentally during echocardiography. This report describes the case of a 68-year-old man, referred to cardiology for multiple masses of the left ventricle and left atrium. The transthoracic echocardiography revealed multiple oscillating masses in the left ventricle and aortic valve, non-mobile mass in the left atrium with severe mitral stenosis and moderate aortic regurgitation. The patient underwent surgical resection of the masses with valve replacements. Histopathologic examination confirmed the diagnosis of CPF in the left ventricle and aortic valve, thrombus in the left atrium.
Cardiac papillary fibroelastomas; Cardiac tumor; Left atrial thrombus; Echocardiography; Cardiac magnetic resonance imaging
A 62-year-old woman with six months history of dizziness was admitted to our hospital. A large mass in the left atrium was detected by transthoracic echocardiography. Coronary angiography showed two feeding arteries from the right coronary artery and left circumflex artery to the left atrium. Chest computed tomography, coronary computed tomographic angiography and contrast echocardiography were performed. Those showed multiple intratumoral neovascularities from surface of the mass. After those examinations, the mass was completely resected. Histopathologic examination confirmed the diagnosis of cardiac myxoma. There was no abnormal remnant mass, based on a follow-up transthoracic echocardiography.
Myxoma; coronary angiography; multiple neovascularity
Necrotizing fasciitis is a life-threatening infectious disease with rapidly progressive involvement of the affected site. Because of the high mortality rate of this disease, early diagnosis, surgical exploration, and administration of appropriate antibiotics are necessary. The present study aimed to further review the changes in the clinical and microbiological characteristics of necrotizing fasciitis using patients' medical records from consecutive databases of 3 hospitals in Korea.
Materials and Methods
In this study, we retrospectively reviewed the medical records of patients with necrotizing fasciitis who were clinically diagnosed between May 2001 and February 2012 in 3 university hospitals in Korea. In total, the data of 83 patients were analyzed, including those of 20 patients from our previous study in 2006. An organism found in a blood culture or surgical specimen was regarded as a causative organism.
Of the 83 patients, 68(81.9%) had community-acquired infections. Ninety microorganism species were indentifed by culture. Streptococcus was the most commonly identified pathogen. Non-fermentative gram-negative bacteria and Candida species have recently emerged, especially in immunocompromised hosts.
Gram-positive organisms are still the most common pathogens of necrotizing fasciitis. However in our study, various gram-negative bacteria with different levels of susceptibility to antibiotics, as well as Candida species, were responsible for the necrotizing fasciitis. Initial empirical antimicrobial agents for necrotizing fasciitis should be considered depending on the individual patient's condition.
Necrotizing fasciitis; Skin and soft tissue infection
During percutaneous coronary intervention, guidewire fractures are very exceptionally encountered in medical practice, but can cause fatal complications such as intracoronary thrombus formation, embolization and perforation. Removal of the remnant segments of guidewire is important for the prognosis. There are several methods being recommended for the treatment of fractured guidewire remnants. However, the best treatment of remnant guidewire filament is still unclear. Herein, we present a case where we did not completely remove remnant guidewire filaments that caused fatal coronary thrombosis.
Percutaneous; Stents; Thrombosis; Complications
The microbiota plays a critical, weight-promoting role in diet-induced obesity (DIO), but the pathways that cause the microbiota to induce weight gain are unknown. We report that mice deficient in lymphotoxin (LT), a key molecule in gut immunity, were resistant to DIO. Ltbr−/− mice differed in microbial community composition compared to their heterozygous littermates, including an overgrowth of segmented filamentous bacteria (SFB). Furthermore, cecal transplantation conferred leanness to germ-free recipients. Housing Ltbr−/− mice with their obese siblings rescued weight gain, demonstrating the communicability of the obese phenotype. Ltbr−/− animals lacked interleukin 23 (IL-23) and IL-22 that can regulate SFB. Mice deficient in these pathways also resisted DIO, demonstrating that intact mucosal immunity guides diet-induced changes to the microbiota to enable obesity.
Pulmonary artery (PA) aneurysm is a rare finding in the thoracic cavity, accompanied by pulmonary hypertension. Clinical presentation of PA aneurysms is usually asymptomatic. The guideline for PA aneurysm treatment is unclear. We report an unusual case of low pressure PA aneurysm associated with atrial septal defect in a 69-year-old man.
Low pressure; Pulmonary artery; Aneurysm; Atrial septal defect
We have designed a five-year multicentre prospective cohort study in women who are both human papillomavirus (HPV)-positive with either atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL) of cervix. This study aimed to analyze the risk of developing a high-grade squamous intraepithelial lesion (HSIL) from either ASCUS or LSIL in HPV-positive women, so called 'progression' rate, to investigate differences in the progression rates according to HPV type-specific infection, and to evaluate the various factors associated with the persistence or clearance of HPV infection in the Korean population. At present, the study protocol composed of cervical cytology, HPV DNA testing, and questionnaire have been conducted actively since the first participant was enrolled in 2010. This study is the first nationwide Korea HPV cohort study. Our data will provide valuable information about not only the ambiguous cytology results of ASCUS and LSIL but also the effect of the specific HPV type and other various factors on the progression to HSIL. Finally, the results of our study will be helpful and applicable to determine the primary cervical cancer prevention strategies.
Cervical intraepithelial neoplasms; Cohort studies; Human papillomavirus; Uterine cervical neoplasms
The possible beneficial effects of chronic low-dose irradiation (LDR) and its mechanism of action in a variety of pathophysiological processes such as cancer are a subject of intense investigation. While animal studies involving long-term exposure to LDR have yielded encouraging results, the influence of LDR at the cellular level has been less well defined. We reasoned that since natural killer (NK) cells constitute an early responder to exogenous stress, NK cells may reveal sentinel alterations in function upon exposure to LDR. When purified NK cells received LDR at 4.2 mGy/h for a total of 0.2 Gy in vitro, no significant difference in cell viability was observed. Likewise, no functional changes were detected in LDR-exposed NK cells, demonstrating that LDR alone was insufficient to generate changes at the cellular level. Nonetheless, significant augmentation of cytotoxic, but not proliferative, function was detected when NK cells were stimulated with low-dose IL-2 prior to irradiation. This enhancement of NK cytotoxicity was not due to alterations in NK-activating receptors, NK1.1, NKG2D, CD69 and 2B4, or changes in the rate of early or late apoptosis. Therefore, LDR, in the presence of suboptimal cytokine levels, can facilitate anti-tumor cytotoxicity of NK cells without influencing cellular proliferation or apoptosis. Whether these results translate to in vivo consequences remains to be seen; however, our data provide initial evidence that exposure to LDR can lead to subtle immune-enhancing effects on NK cells and may explain, in part, the functional basis underlying, diverse beneficial effects seen in the animals chronically exposed to LDR.
Low-dose radiation; natural killer cells; natural cytotoxicity; innate immunity
We tested the efficacy of dual targeting of vascular endothelial growth factor (VEGF) and the alphaVbeta3 integrin in orthotopic mouse models of ovarian cancer.
In the SKOV3ip1 model, both single-agent bevacizumab and etaracizumab reduced tumor growth by 52–63% (p < 0.05), while combined therapy reduced growth by 63–74% compared to either agent alone (p < 0.05). Furthermore, bevacizumab/paclitaxel was superior to paclitaxel alone (weight reduction by 53%, p < 0.05), but etaracizumab/paclitaxel was not. Combining all three agents was more effective than either agent with paclitaxel (p < 0.05). Significantly, both bevacizumab and etaracizumab each sensitized the taxane-resistant SKOV3TRip2 cells to paclitaxel, reducing growth by 56–73% (p < 0.05). Both agents decreased proliferation and microvessel density, and increased apoptosis, alone and in combination with paclitaxel. In the HeyA8 model, there was significantly reduced growth with bevacizumab treatment, but not with etaracizumab, and combination therapy was not superior to bevacizumab alone.
In vivo therapy experiments were conducted in chemo-sensitive (SKOV3ip1, HeyA8) and -resistant (SKOV3TRip2) ovarian cancer models. VEGF was targeted with bevacizumab and alphaVbeta3 with etaracizumab. Mice were treated with each agent alone, together, or in combination with paclitaxel for assessment of tumor growth. Tumor specimens were tested for proliferative index, microvessel density and apoptosis.
Bevacizumab and etaracizumab are more effective in combination than individually in some ovarian cancer models, but not all. Both can sensitize taxane-resistant ovarian cancer cells to paclitaxel, though bevacizumab was superior to etaracizumab in this regard. Further study of this dual anti-angiogenic therapy is warranted.
VEGF; alphaVbeta3; bevacizumab; etaracizumab; ovarian cancer
The cell–microenvironment interaction is critical for cells to perceive environmental cues and co-ordinate signaling cascades to regulate physiological functions. Herein, a soft-lithography technique, micropattern via micromolding in capillaries (MIMIC), is explored to create cell-adhesive micropatterns on glass coverslips. Genetically encoded Src and RhoA fluorescence resonance energy transfer (FRET) biosensors are used to monitor the Src and RhoA activities in nonpatterned cells (stochastically migrating cells, SMCs) and those constrained to grow on micropatterned surfaces (restrictedly migrating cells, RMCs). The results reveal that epidermal growth factor (EGF) induces a decrease of RhoA and an increase of Src activities with biphasic time courses in RMCs. In contrast, the time courses of such activities in SMCs upon EGF stimulation are relatively monophasic. The inhibition of Src activity, actin network, or myosin machinery abolishes the biphasic RhoA response upon EGF stimulation in RMCs. The results indicate that this microenvironment effect on the biphasic RhoA activation in RMCs is mediated by Src and actomyosin machinery. Through the integration of FRET and micropatterning technologies, it is demonstrated that the microenvironment impacts significantly on cell shapes and subsequently the spatiotemporal signaling network of RhoA and Src in living cells. The results help to advance mechanistic understanding of how cells perceive and interpret microenvironments to co-ordinate intracellular molecular signals and ultimately physiological responses.
biosensors; cells; fluorescence resonance; energy transfer; lithography; pattern formation
In the emerging field of RNA-based nanotechnology there is a need for automation of the structure design process. Our goal is to develop computer methods for aiding in this process. Towards that end, we created the RNAJunction database, which is a repository of RNA junctions, i.e. internal, multi-branch and kissing loops with emanating stem stubs, extracted from the larger RNA structures stored in the PDB database. These junctions can be used as building blocks for nanostructures. Two programs developed in our laboratory, NanoTiler and RNA2D3D, can combine such building blocks with idealized fragments of A-form helices to produce desired 3D nanostructures. Initially, the building blocks are treated as rigid objects and the resulting geometry is tested against the design objectives. Experimental data, however, shows that RNA accommodates its shape to the constraints of larger structural contexts. Therefore we are adding analysis of the flexibility of our building blocks to the full design process. Here we present an example of RNA-based nanostructure design, putting emphasis on the need to characterize the structural flexibility of the building blocks to induce ring closure in the automated exploration. We focus on the use of kissing loops (KL) in nanostructure design, since they have been shown to play an important role in RNA self-assembly. By using an experimentally proven system, the RNA tectosquare, we show that considering the flexibility of the KLs as well as distortions of helical regions may be necessary to achieve a realistic design.
RNA; Nanostructure; Design; Modeling; Flexibility; Molecular dynamics