Simultaneous resection of gastric and gallbladder metastasis from renal cell carcinoma treated by laparoscopic and endoscopic cooperative surgery: a case report.

BACKGROUND: Metastases to the stomach or gallbladder from any malignancy is rarely noted, and simultaneous metastases to both organs are atypical. We present a unique case of simultaneous multifocal metastases of the stomach and gallbladder from renal cell carcinoma (RCC). CASE PRESENTATION: The case involved a 60-year-old man, with a past history of RCC (clear cell type, G2, T1b N0 M0 Stage I) treated by a right nephrectomy. Three years after the nephrectomy, a routine gastrointestinal endoscopy found an ulcerative lesion in the greater curvature of the gastric body. The gastric tumor was pathologically proven to be a metastasis from RCC. Furthermore, computed tomography incidentally revealed a mass lesion in the fundus of the gallbladder, which was also diagnosed as a potential metastasis from RCC. As endoscopic ultrasonography of the gastric tumor suggested the tumor potentially invaded to the submucosal layer, gastric wedge resection via a laparoscopic and endoscopic cooperative surgery (LECS) technique was applied to the gastric tumor, and laparoscopic cholecystectomy to the gallbladder tumor was simultaneously performed. Histological examination confirmed that the tumors of the stomach and gallbladder were both metastatic RCC. The hospitalization period after surgery was not eventful, and the patient was discharged on postoperative day 7. Thereafter, the patient required examination every 3 months, did not use anticancer agents, and has survived without relapse to 9 months after the surgery. CONCLUSIONS: For patients with locally resectable RCC metastases, complete metastasectomy may bring long-term tumor control. Moreover, LECS for gastric metastasis is a reasonable approach for minimal invasiveness and an oncologically feasible outcome.

Atomic force microscope as a nano- and micrometer scale biological manipulator: A short review.

The amazing capacity of atomic force microscope to let us touch the molecular and cellular level samples with a sharp probe stimulated its application to bio-medical field among others. In addition to topographical imaging of the sample surface, a direct mechanical manipulation has attracted innovative minds to develop new methodologies aiming at direct handling of proteins, DNA/RNA, and cells. Measurement of their mechanical properties brought about a vivid picture of their physical nature. Direct handling of individual molecules and cells prompted development of nano-medical applications. This short review summarized recent application of AFM for measurement of mechanical properties of biological samples and attempts to perform direct manipulations of nano-medicine.

Membrane wound healing at single cellular level.

We report a nano-technological method of creating a micrometer sized hole on the live cell membrane using atomic force microscope (AFM) and its resealing process at the single cellular level as a model of molecular level wound healing. First, the cell membrane was fluorescently labeled with Kusabira Orange (KO) which was tagged to a lipophilic membrane-sorting peptide. Then a glass bead glued on an AFM cantilever and modified with phospholipase A2 was made to contact the cell membrane. A small dark hole (4-14 µm2 in area) was created on the otherwise fluorescent cell surface often being accompanied by bleb formation. Refilling of holes with KO fluorescence proceeded at an average rate of ~0.014µm2s-1. The fluorescent lumps which initially surrounded the hole were gradually lost. We compared the present result with our previous ones on the repair processes of artificially damaged stress fibers (Graphical Abstract: Figure S2).

Spontaneous Rupture of Renal Metastasis from Hepatocellular Carcinoma.

We report a rare life-threatening case of spontaneous rupture of renal metastasis from hepatocellular carcinoma (HCC) that was managed by emergent transcatheter arterial embolization (TAE). A 76-year-old woman diagnosed with HCC presented with acute back pain in her right side and was transferred to our hospital. Initial enhanced computed tomography revealed retroperitoneal hemorrhage from the right kidney, which was retrospectively diagnosed as a spontaneous rupture of the metastatic renal tumor from the primary HCC. Detailed examination identified an active retroperitoneal hemorrhage from the lesion and the patient's condition became hemodynamically unstable; hence emergent TAE was performed. The hospitalization period after the TAE was uneventful and sorafenib was subsequently administered. Unfortunately, two months after the TAE, the tumor locally progressed within the retroperitoneal space. Tumors were controlled by repeated TAE as the patient did not want to undergo a nephrectomy. Consequently, she survived for more than one year after emergent TAE, exhibiting low levels of tumor marker. After rupture of the metastatic renal HCC, tumors were expected to progress into the retroperitoneal space, and nephrectomy was the next possible radical treatment to offer the best chance of long-term disease control.

Phosphorylated retinoblastoma protein is a potential predictive marker of irinotecan efficacy for colorectal cancer.

Irinotecan has been used in the first-line treatment of metastatic colorectal cancer. However, no clear predictive marker of irinotecan efficacy has been identified. It is controversial whether the response to irinotecan could be predicted by the expression level of topoisomerase-I, a direct target of irinotecan. The present study aimed to identify a feasible predictive marker of irinotecan efficacy. We hypothesized that the efficacy of SN38 (an active metabolite of irinotecan) is related to the cell proliferation and the phosphorylation status of RB in colorectal cancer cells. Indeed, the IC50 of SN38 was positively correlated with the doubling time of each cell line (R2=0.9315). Moreover, the phosphorylation level of RB was related to SN38 sensitivity. Consistent with the in vitro data, colorectal cancer tissues of irinotecan responders showed a significantly higher rate of phosphorylated RB (serine 780) expression using immunohistochemistry (P=0.0006), although a generally used proliferative marker, Ki-67, showed no significance. Finally, we investigated whether the phosphorylation of RB plays a crucial role in the efficacy of irinotecan. To suppress the expression of phosphorylated RB, we performed the knockdown of CDKs, which are known to phosphorylate RB. Intriguingly, the knockdown of both CDK4 and CDK6, but not CDK2, allowed RB to become the most hypophosphorylated form and converted the SN38-sensitive cells to a resistant state. Taking together the above findings from in vitro and clinical research, the immunohistochemistry of phosphorylated RB protein might be feasible to predict the irinotecan efficacy of colorectal cancer in clinical practice.

Spectrin-ankyrin interaction mechanics: A key force balance factor in the red blood cell membrane skeleton.

As major components of red blood cell (RBC) cytoskeleton, spectrin and F-actin form a network that covers the entire cytoplasmic surface of the plasma membrane. The cross-linked two layered structure, called the membrane skeleton, keeps the structural integrity of RBC under drastically changing mechanical environment during circulation. We performed force spectroscopy experiments on the atomic force microscope (AFM) as a means to clarify the mechanical characteristics of spectrin-ankyrin interaction, a key factor in the force balance of the RBC cytoskeletal structure. An AFM tip was functionalized with ANK1-62k and used to probe spectrin crosslinked to mica surface. A force spectroscopy study gave a mean unbinding force of ~30 pN under our experimental conditions. Two energy barriers were identified in the unbinding process. The result was related to the well-known flexibility of spectrin tetramer and participation of ankyrin 1-spectrin interaction in the overall balance of membrane skeleton dynamics.

Subunit unbinding mechanics of dimeric wheat germ agglutinin (WGA) studied by atomic force microscopy.

Wheat germ agglutinin (WGA) is an oligomeric lectin widely used as a model of sugar moieties in biochemistry. Subunit association is important for the crosslinking function of WGA, so we used atomic force microscopy to measure the subunit unbinding force of dimeric WGA. We found that the average unbinding force of dimeric WGA is ∼55 pN at ∼1 nN/s loading rate, whereas this unbinding force is increased at least up to 100 pN when WGA is bound to glycophorin A. Moreover, the dissociation rate constant of WGA was calculated to be 1­2 × 10(−2) s(−1), suggesting that dimer dissociation is relatively fast.

[A long-term survivor of cT4 esophageal carcinoma treated via a multimodal approach - a case report].

The patient was a 53-year-old man whose chief complaint was dysphagia. Pretreatment examination revealed 2 types of locally advanced esophageal squamous cell carcinoma at the middle to lower thoracic esophagus. A computed tomography (CT) scan showed a bulky primary tumor suspicious of aortic invasion and cardiac lymph node metastasis. The pretreatment diagnosis was cT4N2M0, cStageIVa. After induction chemotherapy with 5-fluorouracil (5-FU) and cisplatin (CDDP) (the FP regimen) according to the JCOG9907 regimen, subtotal esophagectomy and 2-field lymphadenectomy with retrosternal stomach roll reconstruction were performed. Intraoperatively, the primary tumor showed extensive and firm adhesion to the aortic wall. The postoperative diagnosis was pT4N0M0, pStageIII, RM1. Postoperative chemoradiotherapy (65 Gy+FP) was performed for the residual tumor at the aortic wall. The patient is alive without recurrences 4 years and 6 months after the esophagectomy. Downstaging of the tumor with induction chemotherapy and effective local control with surgery and postoperative chemoradiotherapy may have contributed to the patient's long-term survival. For multimodal treatment of cT4 esophageal cancer, an effective combination of chemotherapy, surgery, and chemoradiotherapy is essential to improve the treatment outcome.

[Gastric cancer arising from gastric polyps in gardner syndrome - a case report].

The patient was a 48-year-old woman who was diagnosed with early gastric cancer during a long-term follow-up period for Gardner syndrome. Subtotal colectomy for colon leiomyoma was performed when the patient was 22 years old. Partial resection of the ileum was performed for ileum leiomyoma at the age of 27. Total resection of the remaining colon with ileostomy was performed for a pelvic desmoid tumor at the age of 40. In addition, resection of a desmoid tumor of the abdominal wall was performed 8 times in the 25 years since the first operation. During the follow-up for gastric polyps associated with Gardner syndrome, gastric cancer was detected from biopsy specimens of a wide range of the fundus polyps. Endoscopic resection was considered not to be applicable because of the extensive nature of the lesion. Total gastrectomy was also considered not to be applicable because of concerns about short bowel syndrome due to intestinal reconstruction. Therefore, proximal gastrectomy with esophagogastric anastomosis was performed. The pathological diagnosis was 0-IIa, 70 × 44 mm, tub1, m, ly0, v0, n0, PM (-), DM (-), stageIA. The postoperative course was uneventful, and the patient was discharged on postoperative day (POD) 16. We speculate that long-term survival of patients with Gardner syndrome without severe short bowel syndrome might result in carcinogenesis of gastric polyps.

Requirement of LIM domains for the transient accumulation of paxillin at damaged stress fibres.

Cells recognize and respond to changes in intra- and extracellular mechanical conditions to maintain their mechanical homeostasis. Linear contractile bundles of actin filaments and myosin II known as stress fibres (SFs) mediate mechanical signals. Mechanical cues such as excessive stress driven by myosin II and/or external force may damage SFs and induce the local transient accumulation of SF-repair complexes (zyxin and VASP) at the damaged sites. Using an atomic force microscope mounted on a fluorescence microscope, we applied mechanical damage to cells expressing fluorescently tagged cytoskeletal proteins and recorded the subsequent mobilization of SF-repair complexes. We found that a LIM protein, paxillin, transiently accumulated at the damaged sites earlier than zyxin, while paxillin knockdown did not affect the kinetics of zyxin translocation. The C-terminal half of paxillin, comprising four-tandem LIM domains, can still translocate to damaged sites on SFs, suggesting that the LIM domain is essential for the mechanosensory function of paxillin. Our findings demonstrate a crucial role of the LIM domain in mechanosensing LIM proteins.

Prognostic impact of tumor IL-6 expression after preoperative chemoradiotherapy in patients with advanced esophageal squamous cell carcinoma.

Elevated serum interleukin-6 (IL-6) levels have been associated with tumor progression and poor prognosis in patients with esophageal carcinoma. The purpose of the present study was to clarify such a relationship in patients with esophageal squamous cell carcinoma with a focus on the possible influence of chemoradiotherapy (CRT) on tumor IL-6 expression. Data regarding 41 patients with clinical T3-T4 tumors who underwent induction chemoradiotherapy followed by surgery (CRT group) and 60 patients with clinical T1-T4 tumors who underwent surgery alone (Surgery group) between 2001 and 2010, were retrospectively analyzed. Tumor IL-6 expression in resected specimens was evaluated by immunohistochemistry. Tumor IL-6 expression was detected in patients with advanced tumors in the Surgery group (21.1% in p(stage) III-IV vs. 0.2% in p(stage) I-II; 27.8% in pT3-4 vs. 0% in pT1-2), and also correlated with primary tumor progression and surgical curability in the Surgery group. In addition, patients with IL-6-positive tumors had significantly shorter overall survival than those with IL-6-negative tumors in the CRT group, and tumor IL-6 expression had an independent prognostic value in multivariate analysis, whereas no significant difference in overall survival was observed between patients with IL-6-positive and those with IL-6-negative tumors in the Surgery group. These results indicate that pre-treatment tumor IL-6 expression correlates with primary tumor progression, and CRT-induced tumor IL-6 expression predicts poor prognosis.

Overexpression of IL-6 by gene transfer stimulates IL-8-mediated invasiveness of KYSE170 esophageal carcinoma cells.

Interleukin-6 (IL-6) has been associated with disease progression and poor prognosis in esophageal carcinoma. The aim of this study was to investigate the possible influence of IL-6 on the biological activities of esophageal carcinoma cells in terms of invasiveness. The human esophageal carcinoma cell line, KYSE170, was transfected with a plasmid vector expressing IL-6, and a stable transfectant overexpressing IL-6 was established. Invasiveness was evaluated by an invasion assay and compared between IL-6 and control transfectants. The invasiveness of the IL-6 transfectant was significantly higher than that of the control transfectant, and was significantly reduced by IL-6-specific siRNA. In reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, IL-8 expression was significantly higher in the IL-6 transfectant than in the control transfectant, whereas the expression of Hepatocyte growth factor (HGF) and Vascular endothelial growth factor (VEGF) was not different. IL-8 expression in the IL-6 transfectant was significantly inhibited by IL-8-specific siRNA, whereas IL-6 expression was not. In addition, the invasiveness of the IL-6 transfectant was significantly reduced by IL-8-specific siRNA. These results indicate that the overexpression of IL-6 increases the invasiveness of KYSE170 esophageal carcinoma cells and IL-6-induced IL-8 plays a predominant role in increasing invasiveness.

Fabricated cantilever for AFM measurements and manipulations: pre-stress analysis of stress fibers.

The atomic force microscope (AFM) is a highly successful instrument for imaging of nanometer-sized samples and measurement of pico- to nano-Newton forces acting between atoms and molecules, especially in liquid. Generally, commercial AFM cantilevers, which have a sharp tip, are used for AFM experiments. In this review, we introduce micro-fabricated AFM cantilevers and show several applications for cell biology. In manipulation of samples on a cellular scale with a force of tens to hundreds of nano-Newtons, attempts have been made to secure the formation of covalent/non-covalent linkages between the AFM probe and the sample surface. However, present chemistry-based modification protocols of cantilevers do not produce strong enough bonds. To measure the tensile strength and other mechanical properties of actin-based thin filaments in both living and semi-intact fibroblast cells, we fabricated a probe with a hooking function by focused ion beam technology and used it to capture, pull and eventually break a chosen thin filament, which was made visible through fusion with fluorescent proteins. Furthermore, we fabricated a microscoop cantilever specifically designed for pulling a microbead attached to a cell. The microscoop cantilevers can realize high-throughput measurements of cell stiffness.

PSK induces apoptosis through the inhibition of activated STAT3 in human esophageal carcinoma cells.

PSK, a protein-bound polysaccharide, is widely used in Japan as an immunopotentiating biological response modifier for cancer patients. PSK exerts antitumor activities through stimulation of the host's immune response; however, few studies have addressed the direct actions of PSK on tumor cells. Recently, it has been found that STAT3 is aberrantly activated in various types of malignancies, and plays a crucial role in tumor cell proliferation and survival. In the present study, STAT3 was constitutively activated in KYSE170 and TE13 esophageal carcinoma cells, and PSK inhibited proliferation and induced apoptosis in these cells in a dose-dependent manner. Based on these findings, the relationship between STAT3 and apoptosis in these cells was investigated. Results showed that PSK inhibited the expression of activated STAT3 and stimulated the expression of pro-apoptotic Bax in a dose-dependent manner, without affecting the expression of anti-apoptotic Bcl-xL and Mcl-1. These results indicate that PSK may induce apoptosis in esophageal carcinoma cells by inhibiting the expression of activated STAT3.

Interaction between pheromone and its receptor of the fission yeast Schizosaccharomyces pombe examined by a force spectroscopy study.

Interaction between P-factor, a peptide pheromone composed of 23 amino acid residues, and its pheromone receptor, Mam2, on the cell surface of the fission yeast Schizosaccharomyces pombe was examined by an atomic force microscope (AFM). An AFM tip was modified with P-factor derivatives to perform force curve measurements. The specific interaction force between P-factor and Mam2 was calculated to be around 120 pN at a probe speed of 1.74 µm/s. When the AFM tip was modified with truncated P-factor derivative lacking C-terminal Leu, the specific interaction between the tip and the cell surface was not observed. These results were also confirmed with an assay system using a green fluorescent protein (GFP) reporter gene to monitor the activation level of signal transduction following the interaction of Mam2 with P-factor.

Forced extension of delipidated red blood cell cytoskeleton with little indication of spectrin unfolding.

Force-extension curves obtained on intact human red blood cells (RBC) were compared with those of delipidated RBCs to assess the contribution of cytoskeletal flexibility to the extensibility of the intact membrane skeleton. The RBCs were first delipidated by treatment with phospholipase A2; tensile properties of the exposed cytoskeletal structures were measured using an atomic force microscope (AFM). The AFM probes were modified either with the Band 3 specific lectin, concanavalin A, (Con A) or anti-F-actin antibody, to localize the point of interaction between the probe and the cytoskeleton. Extension of the spectrin-based cytoskeleton reached up to 2-3 µm with a force less than 70 pN without showing any force peaks before the final rupture of the adhesive bonds. Our interpretation of the result is that the spectrin-based network was slack enough to allow the observed degree of extension without unfolding the tetrameric spectrin molecules. The force-extension curves obtained either on Band 3-ankyrin loci or on junction nodes of the cytoskeleton were not significantly different. Experimental results were verified by computer simulation of pulling mechanics of a network model of the RBC cytoskeleton. Our experimental results are also in agreement with the theoretical prediction of Mirijanian and Voth [2008; Proc Natl Acad Sci USA 105:1204-1208].

Nonlinear displacement of ventral stress fibers under externally applied lateral force by an atomic force microscope.

Actin-based stress fibers (SFs) have fundamental importance in the maintenance of mechanical stability of living cells. Several in vitro measurements of their elastic properties have therefore been made, but direct mechanical manipulation of individual SFs in vivo for the determination of their mechanical properties has not been attempted. No less important is a search for the possible formation of a global mechanical network involving SFs and other intracellular filamentous components. In this article, we present an application of atomic force microscopy to probe into a live cell and laterally push selected SFs in a fibroblast cells (VNOf 06 fibroblast-like cells derived from rat vomeronasal tissue) transfected with a green fluorescent protein-ß-actin gene. The transfected cells were transferred to a serum-depleted medium before the atomic force microscope manipulation. The lateral displacement of the SFs under a point loading condition recorded on a fluorescence microscope revealed both linear and nonlinear displacements against the contour distance from the point of force loading. The nonlinear displacements of the SFs were attributed to their association with a cortical actomyosin-cell membrane complex that effectively pulled them back as a 2D thin plate.

Direct detection of cellular adaptation to local cyclic stretching at the single cell level by atomic force microscopy.

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope. The cell tension initially increased in response to the tensile stress and then decreased within ∼1 min as a result of viscoelastic properties of the cell. This relaxation was followed by a gradual increase in tension extending over several minutes. The slow recovery of tension ceased after several cycles of force application. This tension-recovering activity was inhibited when cells were treated with cytochalasin D, an inhibitor of actin polymerization, or with (-)-blebbistatin, an inhibitor of myosin II ATPase activity, suggesting that the activity was driven by actin-myosin interaction. To our knowledge, this is the first quantitative analysis of cellular mechanical properties during the process of adaptation to locally applied cyclic external force.