Structure-function analysis of the SHOC2-MRAS-PP1C holophosphatase complex.

Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .

Image quality and lesion detectability of deep learning-accelerated T2-weighted Dixon imaging of the cervical spine.

OBJECTIVES: To validate the subjective image quality and lesion detectability of deep learning-accelerated Dixon (DL-Dixon) imaging of the cervical spine compared with routine Dixon imaging. MATERIALS AND METHODS: A total of 50 patients underwent sagittal routine Dixon and DL-Dixon imaging of the cervical spine. Acquisition parameters were compared and non-uniformity (NU) values were calculated. Two radiologists independently assessed the two imaging methods for subjective image quality and lesion detectability. Interreader and intermethod agreements were estimated with weighted kappa values. RESULTS: Compared with the routine Dixon imaging, the DL-Dixon imaging reduced the acquisition time by 23.76%. The NU value is slightly higher in DL-Dixon imaging (p value: 0.015). DL-Dixon imaging showed superior visibility of all four anatomical structures (spinal cord, disc margin, dorsal root ganglion, and facet joint) for both readers (p value: < 0.001 ~ 0.002). The motion artifact scores were slightly higher in the DL-Dixon images than in routine Dixon images (p value = 0.785). Intermethod agreements were almost perfect for disc herniation, facet osteoarthritis, uncovertebral arthritis, central canal stenosis (κ range: 0.830 ~ 0.980, all p values < 0.001) and substantial to almost perfect for foraminal stenosis (κ = 0.955, 0.705 for each reader). There was an improvement in the interreader agreement of foraminal stenosis by DL-Dixon images, from moderate to substantial agreement. CONCLUSION: The DLR sequence can substantially decrease the acquisition time of the Dixon sequence with subjective image quality at least as good as the conventional sequence. And no significant differences in lesion detectability were observed between the two sequence types.

Minimally invasive surgery versus open surgery in high-risk histologic endometrial cancer patients: A meta-analysis.

OBJECTIVE: To compare the effects of minimally invasive surgery (MIS) and open surgery (OPS) on the risk of recurrence and mortality in patients with endometrial cancer (EC) of high-risk histology (grade 3 endometrioid adenocarcinoma, papillary serous carcinoma [PS], clear cell carcinoma [CC], and carcinosarcoma) using meta-analysis. MATERIAL AND METHODS: We systematically reviewed published studies comparing MIS and OPS in EC patients with high-risk histology until January 2022. The endpoints were recurrence and mortality rate. Study design features that may have affected participant selection, recurrence/death detection, and manuscript publication were assessed. For pooled estimates of the effect of MIS on recurrence/mortality, the random- or fixed-effects meta-analytical models were used after assessing the cross-study heterogeneity. RESULT: Nine observational studies (eight retrospective and one prospective) fulfilled our search criteria (MIS, 8877 patients; OPS, 5751 patients). The fixed-effects model-based meta-analysis indicated that MIS did not significantly increase the risk of recurrence (hazard ratio [HR], 0.86; 95% confidence interval [CI], 0.71-1.05; p = 0.13) and mortality (HR, 0.86; 95% CI, 0.79-0.93; p < 0.001) when compared with OPS. This pattern was also observed in the subgroup analyses based on the stage (early stage vs. all stage), histology (PS and CC), and MIS type (laparoscopy vs. robotic). There was no evidence of publication bias. CONCLUSION: This meta-analysis of observational studies revealed that MIS did not compromise the prognosis of EC patients with high-risk histology. Well-designed randomized controlled trials could verify the results of this uncommon but deadly tumor.

Retreatment with progestin for recurrence after complete response with fertility-sparing treatment in patients with endometrial cancer.

OBJECTIVE: To assess the outcomes of retreatment using progestin for recurrence after a complete response with fertility-sparing treatment in patients with early endometrial cancer. METHODS: We retrospectively reviewed the data of patients with presumed stage IA, grade 1 endometrioid endometrial cancer who developed intra-uterine recurrence after a complete response with fertility-sparing treatment using progestin. Oncological and pregnancy outcomes were analyzed after repeated fertility-sparing treatment. Logistic and Cox regression analyses were performed to analyze the prognostic factors associated with a complete response with secondary fertility-sparing treatment and recurrence-free survival after secondary fertility-sparing treatment, respectively. RESULTS: Fifty patients with a median age of 31 years (range 23-40) underwent secondary fertility-sparing treatment. With a median secondary progestin treatment duration of 9 months (range 3-55), the complete response rate was 78% (39/50) and no patients had extra-uterine spread of disease. Among the 26 (67%) patients who attempted to conceive after complete response, 10 became pregnant (3 spontaneous abortions, 7 live births). Eighteen (46.1%) patients had a second recurrence, with a median recurrence-free survival after secondary fertility-sparing treatment of 14 months (range 3-36); 15 patients received tertiary fertility-sparing treatment and nine (60%) achieved a complete response. Polycystic ovary on ultrasound (OR 5.82, 95% CI 1.1 to 30.6, p=0.037) was associated with an increased complete response rate with secondary fertility-sparing treatment. Multivariable analysis revealed that recurrence-free survival after initial hormonal treatment >6 months (HR 0.11, 95% CI 0.02 to 0.51, p=0.005) and pregnancy after secondary fertility-sparing treatment (HR 0.27, 95% CI 0.08 to 0.98; p=0.047) were significantly associated with longer recurrence-free survival after secondary fertility-sparing treatment. CONCLUSIONS: Repeated progestin treatment was associated with a 78% response rate and it was safe in patients with intra-uterine recurrent endometrial cancer. Thus, it might help preserve fertility after first and second recurrences.

Zileuton Alleviates Radiation-Induced Cutaneous Ulcers via Inhibition of Senescence-Associated Secretory Phenotype in Rodents.

Radiation-induced cutaneous ulcers are a challenging medical problem for patients receiving radiation therapy. The inhibition of cell senescence has been suggested as a prospective strategy to prevent radiation ulcers. However, there is no effective treatment for senescent cells in radiation ulcers. In this study, we investigated whether zileuton alleviated radiation-induced cutaneous ulcer by focusing on cell senescence. We demonstrate increased cell senescence and senescence-associated secretory phenotype (SASP) in irradiated dermal fibroblasts and skin tissue. The SASP secreted from senescent cells induces senescence in adjacent cells. In addition, 5-lipoxygenase (5-LO) expression increased in irradiated dermal fibroblasts and skin tissue, and SASP and cell senescence were regulated by 5-LO through p38 phosphorylation. Finally, the inhibition of 5-LO following treatment with zileuton inhibited SASP and mitigated radiation ulcers in animal models. Our results demonstrate that inhibition of SASP from senescent cells by zileuton can effectively mitigate radiation-induced cutaneous ulcers, indicating that inhibition of 5-LO might be a viable strategy for patients with this condition.

Potassium channel selectivity filter dynamics revealed by single-molecule FRET.

Potassium (K) channels exhibit exquisite selectivity for conduction of K+ ions over other cations, particularly Na+. High-resolution structures reveal an archetypal selectivity filter (SF) conformation in which dehydrated K+ ions, but not Na+ ions, are perfectly coordinated. Using single-molecule FRET (smFRET), we show that the SF-forming loop (SF-loop) in KirBac1.1 transitions between constrained and dilated conformations as a function of ion concentration. The constrained conformation, essential for selective K+ permeability, is stabilized by K+ but not Na+ ions. Mutations that render channels nonselective result in dilated and dynamically unstable conformations, independent of the permeant ion. Further, while wild-type KirBac1.1 channels are K+ selective in physiological conditions, Na+ permeates in the absence of K+. Moreover, whereas K+ gradients preferentially support 86Rb+ fluxes, Na+ gradients preferentially support 22Na+ fluxes. This suggests differential ion selectivity in constrained versus dilated states, potentially providing a structural basis for this anomalous mole fraction effect.

Optimal duration of fertility-sparing hormonal treatment for early-stage endometrioid endometrial cancer.

OBJECTIVES: To analyze the oncologic outcomes of long-term fertility-sparing treatment (FST) in patients with early-stage endometrial cancer (EC) and to determine the optimal duration of FST that would not hamper survival outcomes. METHODS: Patients undergoing FST for presumed stage IA, grade 1 EC between 2005 and 2018 were retrospectively analyzed. Oncologic outcomes were compared between the group with ≤6 months of FST and the group with >6 months of FST. Segmented regression analysis was used to estimate the dynamic changes in cumulative complete response (CR) rates according to FST duration. RESULTS: A total of 122 patients received oral progestin, with concurrent levonorgestrel-releasing intrauterine device use in 108 (88.5%) and 105 (86.1%) achieved CR with a median time to achieve CR of 10 (3-42) months. Of the patients not achieving CR at 6 months of FST, 95.1% (78/82) continued further FST. The overall CR rate (88.9% [32/36] vs. 84.9% [73/86], P = 0.436] was not significantly different between the groups with ≤6 and > 6 months of FST. The changes in cumulative CR rates were significantly different between the two segments divided by 15 months from the initial FST (P = 0.0015, segmented regression analysis). The overall progressive disease (PD) rate was 3.3% (4/122), and PD was first detected during 9-12 months of FST. CONCLUSION: Patients not achieving CR and not showing PD at 6 months of FST could continue further FST. If disease progression is excluded, 15 months of FST can be considered as the cutoff for the optimal FST duration.

Peripheral nerve sheath tumor: differentiation of malignant from benign tumors with conventional and diffusion-weighted MRI.

OBJECTIVES: To evaluate potential of conventional MRI and diffusion-weighted imaging (DWI) for differentiating malignant from benign peripheral nerve sheath tumors (PNSTs). METHODS: Eighty-seven cases of malignant or benign PNSTs in the trunk or extremities that underwent conventional MRI with contrast enhancement, DWI, and pathologic confirmation between Sep. 2014 and Dec. 2017 were identified. Of these, 55 tumors of uncertain nature on MRI were included. Tumor size, signal, and morphology were reviewed on conventional MRI, and apparent diffusion coefficient (ADC) values of solid enhancing portions were measured from DWI. Patient demographics, MRI features, and ADC values were compared between benign and malignant tumors, and robust imaging findings for malignant peripheral nerve sheath tumors (MPNSTs) were identified using multivariable models. RESULTS: A total of 55 uncertain tumors consisted of 18 malignant and 37 benign PNSTs. On MRI, tumor size, margin, perilesional edema, and presence of split fat, fascicular, and target signs were significantly different between groups (p < 0.05), as were mean and minimum ADC values (p = 0.002, p < 0.0001). Most inter-reader agreement was moderate to excellent (κ value, 0.45-1.0). The mean ADC value and absence of a split fat sign were identified as being associated with MPNSTs (odds ratios = 13.19 and 25.67 for reader 1; 49.05 and 117.91 for reader 2, respectively). The C-indices obtained by combining these two findings were 0.90 and 0.95, respectively. CONCLUSIONS: Benign and malignant PNSTs showed different features on MRI and DWI. A combination of mean ADC value and absence of split fat was excellent for discriminating malignant from benign PNSTs. KEY POINTS: • It is important to distinguish between malignant peripheral nerve sheath tumors (MPNSTs) and benign peripheral nerve sheath tumors (BPNSTs) to ensure an appropriate treatment plan. • On conventional MRI and diffusion-weighted imaging (DWI), MPNSTs and BPNSTs showed significant differences in tumor size, margin, presence of perilesional edema, and absence of split fat, fascicular, and target signs. • Absence of a split fat sign and mean apparent diffusion coefficient (ADC) values were robust imaging findings distinguishing MPNSTs from BPNSTs, with a C-index of > 0.9.

Computed tomography arthrography of the shoulder with tin filter-based spectral shaping at 100 kV and 140 kV.

BACKGROUND: Tin filter-based spectral shaping has been used for low-dose and ultra-low-dose computed tomography (CT) in several body parts. However, studies of shoulder CT arthrography with spectral shaping are limited. PURPOSE: To investigate image quality and radiation dose of shoulder CT arthrography with tin filter-based spectral shaping at 100 kV (Sn 100 kV) and 140 kV (Sn 140 kV) in comparison with the conventional protocol. MATERIAL AND METHODS: Ninety-nine shoulder CT arthrographies with protocols of Sn 100 kV (n = 32), Sn 140 kV (n = 25), and conventional 120 kV (n = 42) were retrospectively evaluated. Qualitative image quality, CT attenuations of intra-articular contrast mixture and tissues, background noise, contrast-to-noise ratios (CNRs), and figures of merit were assessed. Radiation doses were compared. RESULTS: CT arthrographies with Sn 100 kV and Sn 140 kV yielded approximately 70% and 60% radiation dose reduction, respectively, compared with the conventional 120 kV (P < 0.001). Qualitative image noise and quantitative background noise of Sn 100 kV and Sn 140 kV were significantly less than those of the conventional protocol. Qualitative image contrast, CT attenuations of intra-articular contrast mixture and tissues, and CNRs for Sn 100 were similar to those of the conventional 120 kV. However, Sn 140 kV showed significantly lower qualitative contrast and CNRs than 120 kV. Sn 100 kV was the most dose efficient among the three protocols. CONCLUSION: Shoulder CT arthrography with Sn 100 kV substantially reduced radiation dose and image noise and maintained image contrast, compared with the conventional protocol.

Sarcopenia as a Predictor of Prognosis in Early Stage Ovarian Cancer.

BACKGROUND: To identify sarcopenia as a predictive prognostic factor of ovarian cancer in terms of survival outcome in patients with early-stage ovarian cancer. METHODS: Data of Konkuk University Medical Center from March 2002 to December 2017 were reviewed retrospectively. Eighty-two patients who underwent surgery due to early-stage (International Federation of Gynecology and Obstetrics stage I/II) ovarian cancer and had computed tomography (CT) images taken at the initial diagnosis were included. The initial CT scan images were analyzed with SliceOmatic software (TomoVision). A sarcopenia cutoff value was defined as a skeletal muscle index of ≤ 38.7 cm²/m². Overall survival (OS) times were compared according to the existence of sarcopenia, and subgroup analyses were performed. RESULTS: A Kaplan-Meier analysis showed a significant survival disadvantage for patients with early-stage ovarian cancer when they had sarcopenia (P < 0.001; log-rank test). Sarcopenia remained a significant prognostic factor for OS in early-stage ovarian cancer, in a Cox proportional hazards model regression analysis (HR, 21.9; 95% CI, 2.0-199.9; P = 0.006). CONCLUSION: This study demonstrated that sarcopenia was predictive of OS in patients with early-stage ovarian cancer. Further prospective studies with a larger number of patients are warranted to determine the extent to which sarcopenia can be used as a prognostic factor in ovarian cancer.

Atorvastatin Inhibits Endothelial PAI-1-Mediated Monocyte Migration and Alleviates Radiation-Induced Enteropathy.

Intestinal injury is observed in cancer patients after radiotherapy and in individuals exposed to radiation after a nuclear accident. Radiation disrupts normal vascular homeostasis in the gastrointestinal system by inducing endothelial damage and senescence. Despite advances in medical technology, the toxicity of radiation to healthy tissue remains an issue. To address this issue, we investigated the effect of atorvastatin, a commonly prescribed hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor of cholesterol synthesis, on radiation-induced enteropathy and inflammatory responses. We selected atorvastatin based on its pleiotropic anti-fibrotic and anti-inflammatory effects. We found that atorvastatin mitigated radiation-induced endothelial damage by regulating plasminogen activator inhibitor-1 (PAI-1) using human umbilical vein endothelial cells (HUVECs) and mouse model. PAI-1 secreted by HUVECs contributed to endothelial dysfunction and trans-endothelial monocyte migration after radiation exposure. We observed that PAI-1 production and secretion was inhibited by atorvastatin in irradiated HUVECs and radiation-induced enteropathy mouse model. More specifically, atorvastatin inhibited PAI-1 production following radiation through the JNK/c-Jun signaling pathway. Together, our findings suggest that atorvastatin alleviates radiation-induced enteropathy and supports the investigation of atorvastatin as a radio-mitigator in patients receiving radiotherapy.

Second-look arthroscopic findings and clinical outcomes after management of intra-articular lesions in acute ankle fractures with arthroscopy.

BACKGROUND: Anatomically surgical reduction of ankle fractures does not always result in a clinically favorable outcome. Arthroscopic examination combined with treatment of intra-articular lesion may related to clinical outcomes OBJECTIVES: The purpose of the present study was to review initial and second look arthroscopic finding of acute ankle fracture and to evaluate clinical outcomes. RESULTS: Lauge-Hansen classification system of ankle fractures included supination-external rotation type (n = 24), supination-adduction type (n = 3), pronation-external rotation type (n = 7), and pronation-abduction type (n = 6), total 40 ankles. Osteochondral lesions were found in 25 ankles (62%) with an initial arthroscopic finding of acute ankle fracture. Newly discovered chondral lesions in secondary arthroscopy were found in 17 cases. According to the Ferkel and Cheng staging at secondary arthroscopy, 4 of 25 ankles with osteochondral lesions of the talus were deteriorating (more than stage D). In terms of ICRS overall repair grades, 5 ankles (20%) were abnormal (grade III). Diffuse synovitis and arthrofibrosis were found in 12 and 7 ankles, respectively, in secondary arthroscopy, and correlations were found between AOFAS scores, VAS and intra-articular lesions. CONCLUSION: Second-look arthroscopic examination combined with treatment of intra-articular lesion such as arthrofibrosis and osteochondral lesion arising from ankle fracture surgery may consider to improve clinical outcomes.

Platelet-rich plasma improves the therapeutic efficacy of mesenchymal stem cells by enhancing their secretion of angiogenic factors in a combined radiation and wound injury model.

Delayed wound healing after radiation exposure can cause serious cutaneous damage, and its treatment is a major clinical challenge. Although mesenchymal stem cells (MSCs) have emerged as a promising therapeutic agent in regenerative medicine, they alone do not produce satisfactory effects in a combined radiation and wound injury (CRWI) model. Here, we investigated the therapeutic effect of combined umbilical cord blood-derived (UCB)-MSCs and platelet-rich plasma (PRP) treatment on wound healing in a CRWI mouse model. First, we assessed the release of cytokines from UCB-MSCs cultured with PRP and observed changes in the expression of angiogenic factors. The angiogenic paracrine factors from UCB-MSCs cultured with PRP were assessed in human umbilical vein endothelial cells (HUVECs). To assess therapeutic efficacy, UCB-MSCs and PRP were topically implanted into a CRWT mouse model. Vascular endothelial growth factor (VEGF), a pro-angiogenic growth factor, urokinase-type plasminogen activator and contributor to VEGF-induced signalling were more highly expressed in conditioned media of UCB-MSCs cultured with PRP than in that of UCB-MSCs alone. Furthermore, conditioned media of UCB-MSCs cultured with PRP increased the formation of tube-like structures in HUVECs. Co-treatment of UCB-MSCs and PRP in a CRWI mouse model increased the wound closure rate and angiogenesis compared with an untreated irradiated group. Moreover, increased expression of VEGF and CD31 were observed in the wound tissue of co-treated mice compared with untreated irradiated mice. PRP stimulates the release of angiogenic factors from UCB-MSCs, and combined therapy of UCB-MSCs and PRP improves regeneration efficacy by enhancing angiogenesis in a CRWI model.

Evaluation of the radiation response and regenerative effects of mesenchymal stem cell-conditioned medium in an intestinal organoid system.

Intestinal organoids have recently emerged as an in vitro model relevant to the gut system owing to their recapitulation of the native intestinal epithelium with crypt-villus architecture. However, it is unclear whether intestinal organoids reflect the physiology of the in vivo stress response. Here, we systemically investigated the radiation response in organoids and animal models using mesenchymal stem cell-conditioned medium (MSC-CM), which contains secreted paracrine factors. Irradiated organoids exhibited sequential induction of viability loss and regrowth after irradiation (within 12 days), similar to the response of the native intestinal epithelium. Notably, treatment with MSC-CM facilitated the reproliferation of intestinal stem cells (ISCs) and restoration of damaged crypt-villus structures in both models. Furthermore, Wnt/Notch signaling pathways were commonly upregulated by MSC-CM, but not radiation, and pharmacologically selective inhibition of Wnt or Notch signaling attenuated the enhanced recovery of irradiated organoids, with increases in ISCs, following MSC-CM treatment. Interestingly, the expression of Wnt4, Wnt7a, and active ß-catenin was increased, but not notch family members, in MSC-CM-treated organoid after irradiation. Treatment of recombinant mouse Wnt4 and Wnt7a after irradiation improved to some extent intestinal epithelial regeneration both in vitro and in vivo. Overall, these results suggested that intestinal organoids recapitulated the physiological stress response of the intestinal epithelium in vivo. Thus, our findings provided important insights into the physiology of intestinal organoids and may contribute to the development of strategies to enhance the functional maturation of engineered organoids.

Vascular Calcification-New Insights Into Its Mechanism.

Vascular calcification (VC), which is categorized by intimal and medial calcification, depending on the site(s) involved within the vessel, is closely related to cardiovascular disease. Specifically, medial calcification is prevalent in certain medical situations, including chronic kidney disease and diabetes. The past few decades have seen extensive research into VC, revealing that the mechanism of VC is not merely a consequence of a high-phosphorous and -calcium milieu, but also occurs via delicate and well-organized biologic processes, including an imbalance between osteochondrogenic signaling and anticalcific events. In addition to traditionally established osteogenic signaling, dysfunctional calcium homeostasis is prerequisite in the development of VC. Moreover, loss of defensive mechanisms, by microorganelle dysfunction, including hyper-fragmented mitochondria, mitochondrial oxidative stress, defective autophagy or mitophagy, and endoplasmic reticulum (ER) stress, may all contribute to VC. To facilitate the understanding of vascular calcification, across any number of bioscientific disciplines, we provide this review of a detailed updated molecular mechanism of VC. This encompasses a vascular smooth muscle phenotypic of osteogenic differentiation, and multiple signaling pathways of VC induction, including the roles of inflammation and cellular microorganelle genesis.

Pravastatin Alleviates Radiation Proctitis by Regulating Thrombomodulin in Irradiated Endothelial Cells.

Although radiotherapy plays a crucial in the management of pelvic tumors, its toxicity on surrounding healthy tissues such as the small intestine, colon, and rectum is one of the major limitations associated with its use. In particular, proctitis is a major clinical complication of pelvic radiotherapy. Recent evidence suggests that endothelial injury significantly affects the initiation of radiation-induced inflammation. The damaged endothelial cells accelerate immune cell recruitment by activating the expression of endothelial adhesive molecules, which participate in the development of tissue damage. Pravastatin, a cholesterol lowering drug, exerts persistent anti-inflammatory and anti-thrombotic effects on irradiated endothelial cells and inhibits the interaction of leukocytes and damaged endothelial cells. Here, we aimed to investigate the effects of pravastatin on radiation-induced endothelial damage in human umbilical vein endothelial cell and a murine proctitis model. Pravastatin attenuated epithelial damage and inflammatory response in irradiated colorectal lesions. In particular, pravastatin improved radiation-induced endothelial damage by regulating thrombomodulin (TM) expression. In addition, exogenous TM inhibited leukocyte adhesion to the irradiated endothelial cells. Thus, pravastatin can inhibit endothelial damage by inducing TM, thereby alleviating radiation proctitis. Therefore, we suggest that pharmacological modulation of endothelial TM may limit intestinal inflammation after irradiation.

Polyamines and potassium channels: A 25-year romance.

Potassium channels that exhibit the property of inward rectification (Kir channels) are present in most cells. Cloning of the first Kir channel genes 25 years ago led to recognition that inward rectification is a consequence of voltage-dependent block by cytoplasmic polyamines, which are also ubiquitously present in animal cells. Upon cellular depolarization, these polycationic metabolites enter the Kir channel pore from the intracellular side, blocking the movement of K+ ions through the channel. As a consequence, high K+ conductance at rest can provide very stable negative resting potentials, but polyamine-mediated blockade at depolarized potentials ensures, for instance, the long plateau phase of the cardiac action potential, an essential feature for a stable cardiac rhythm. Despite much investigation of the polyamine block, where exactly polyamines get to within the Kir channel pore and how the steep voltage dependence arises remain unclear. This Minireview will summarize current understanding of the relevance and molecular mechanisms of polyamine block and offer some ideas to try to help resolve the fundamental issue of the voltage dependence of polyamine block.

Hyaluronic acid synthase 2 promotes malignant phenotypes of colorectal cancer cells through transforming growth factor beta signaling.

Hyaluronic acid synthase 2 (HAS2) is suggested to play a critical role in malignancy and is abnormally expressed in many carcinomas. However, its role in colorectal cancer (CRC) malignancy and specific signaling mechanisms remain obscure. Here, we report that HAS2 was markedly increased in both CRC tissue and malignant CRC cell lines. Depletion of HAS2 in HCT116 and DLD1 cells, which express high levels of HAS2, critically increased sensitivity of radiation/oxaliplatin-mediated apoptotic cell death. Moreover, downregulation of HAS2 suppressed migration, invasion and metastasis in nude mice. Conversely, ectopic overexpression of HAS2 in SW480 cells, which express low levels of HAS2, showed the opposite effect. Notably, HAS2 loss- and gain-of-function experiments revealed that it regulates CRC malignancy through TGF-ß expression and SMAD2/Snail downstream components. Collectively, our findings suggest that HAS2 contributes to malignant phenotypes of CRC, at least partly, through activation of the TGF-ß signaling pathway, and shed light on the novel mechanisms behind the constitutive activation of HAS2 signaling in CRC, thereby highlighting its potential as a therapeutic target.

Effects of metformin and phenformin on apoptosis and epithelial-mesenchymal transition in chemoresistant rectal cancer.

Recurrence and chemoresistance in colorectal cancer remain important issues for patients treated with conventional therapeutics. Metformin and phenformin, previously used in the treatment of diabetes, have been shown to have anticancer effects in various cancers, including breast, lung and prostate cancers. However, their molecular mechanisms are still unclear. In this study, we examined the effects of these drugs in chemoresistant rectal cancer cell lines. We found that SW837 and SW1463 rectal cancer cells were more resistant to ionizing radiation and 5-fluorouracil than HCT116 and LS513 colon cancer cells. In addition, metformin and phenformin increased the sensitivity of these cell lines by inhibiting cell proliferation, suppressing clonogenic ability and increasing apoptotic cell death in rectal cancer cells. Signal transducer and activator of transcription 3 and transforming growth factor-ß/Smad signaling pathways were more activated in rectal cancer cells, and inhibition of signal transducer and activator of transcription 3 expression using an inhibitor or siRNA sensitized rectal cancer cells to chemoresistant by inhibition of the expression of antiapoptotic proteins, such as X-linked inhibitor of apoptosis, survivin and cellular inhibitor of apoptosis protein 1. Moreover, metformin and phenformin inhibited cell migration and invasion by suppression of transforming growth factor ß receptor 2-mediated Snail and Twist expression in rectal cancer cells. Therefore, metformin and phenformin may represent a novel strategy for the treatment of chemoresistant rectal cancer by targeting signal transducer and activator of transcription 3 and transforming growth factor-ß/Smad signaling.

The PP2AB56 phosphatase promotes the association of Cdc20 with APC/C in mitosis.

PP2A comprising B56 regulatory subunit isoforms (PP2AB56) is a serine/threonine phosphatase essential for mitosis. At the kinetochore, PP2AB56 both stabilizes microtubule binding and promotes silencing of the spindle assembly checkpoint (SAC) through its association with the SAC protein BubR1. Cells depleted of the B56 regulatory subunits of PP2A are delayed in activation of Cdc20-containing APC/C (APC/CCdc20), which is an essential step for mitotic exit. It has been hypothesized that this delay arises from increased production of the mitotic checkpoint complex (MCC), an APC/CCdc20 inhibitor formed at unattached kinetochores through SAC signaling. In contrast to this prediction, we show that depletion of B56 subunits does not increase the amount or stability of the MCC. Rather, delays in APC/CCdc20 activation in B56-depleted cells correlate with impaired Cdc20 binding to APC/C. Stimulation of APC/CCdc20 assembly does not require binding between PP2AB56 and BubR1, and thus this contribution of PP2AB56 towards mitotic exit is distinct from its functions at kinetochores. PP2AB56 associates with APC/C constitutively in a BubR1-independent manner. A mitotic phosphorylation site on Cdc20, known to be a substrate of PP2AB56, modulates APC/CCdc20 assembly. These results elucidate the contributions of PP2AB56 towards completion of mitosis.