ß-catenin inhibitor ICG-001 suppress cell cycle progression and induce autophagy in endometrial cancer cells.

The incidence of endometrial cancer has been rising in recent years. Gene mutation and high protein expression of ß-catenin are commonly detected in endometrioid endometrial cancer. ICG-001 is a ß-catenin inhibitor via blocking the complex formation of ß-catenin and cAMP response element-binding protein (CREB)-binding protein (CBP). This study aims to investigate the effect of ICG-001 on endometrial cancer inhibition. First, endometrial carcinoma patient-derived xenograft (PDX)-derived organoids and primary cells were used to verify the inhibiting ability of ICG-001 on endometrial cancer. Furthermore, endometrial cancer cell lines were used to investigate the anticancer mechanism of ICG-001. Using MTT assay and tumor spheroid formation assay, ICG-001 significantly reduced the cell viability of HEC-59 and HEC-1A cells. ICG-001 enhanced cisplatin-mediated cytotoxicity. ICG-001 decreased cancer stem cell sphere formation. ICG-001 decreased the protein expressions of CD44, hexokinase 2 (HK2), and cyclin A. ICG-001 lowered the cell cycle progression by flow cytometer analysis. Autophagy, but no apoptosis, was activated by ICG-001 in endometrial cancer cells. Autophagy inhibition by ATG5 silencing enhanced ICG-001-mediated suppression of cell viability, tumor spheroid formation, and protein expression of cyclin A and CD44. This study clarified the mechanism and revealed the clinical potential of ICG-001 against endometrial cancer.

Suppression of PI3K/Akt/mTOR/c-Myc/mtp53 Positive Feedback Loop Induces Cell Cycle Arrest by Dual PI3K/mTOR Inhibitor PQR309 in Endometrial Cancer Cell Lines.

Gene mutations in PIK3CA, PIK3R1, KRAS, PTEN, and PPP2R1A commonly detected in type I endometrial cancer lead to PI3K/Akt/mTOR pathway activation. Bimiralisib (PQR309), an orally bioavailable selective dual inhibitor of PI3K and mTOR, has been studied in preclinical models and clinical trials. The aim of this study is to evaluate the anticancer effect of PQR309 on endometrial cancer cells. PQR309 decreased cell viability in two-dimensional and three-dimensional cell culture models. PQR309 induced G1 cell cycle arrest and little cell death in endometrial cancer cell lines. It decreased CDK6 expression and increased p27 expression. Using the Proteome Profiler Human XL Oncology Array and Western blot assay, the dual inhibitor could inhibit the expressions of c-Myc and mtp53. KJ-Pyr-9, a c-Myc inhibitor, was used to prove the role of c-Myc in endometrial cancer survival and regulating the expression of mtp53. Knockdown of mtp53 lowered cell proliferation, Akt/mTOR pathway activity, and the expressions of c-Myc. mtp53 silence enhanced PQR309-inhibited cell viability, spheroid formation, and the expressions of p-Akt, c-Myc, and CDK6. This is the first study to reveal the novel finding of the PI3K/mTOR dual inhibitor in lowering cell viability by abolishing the PI3K/Akt/mTOR/c-Myc/mtp53 positive feedback loop in endometrial cancer cell lines.

D-Methionine Ameliorates Cisplatin-Induced Muscle Atrophy via Inhibition of Muscle Degradation Pathway.

Cisplatin induces anorexia, weight loss, loss of adipose tissue, skeletal muscle atrophy, and serious adverse effects that can cause premature termination of chemotherapy. The aim of this study was to use an animal model to assess cisplatin therapy (3 cycles) with and without d-methionine to investigate its protective effects on cisplatin-induced anorexia and skeletal muscle wasting. Wistar rats were divided into 3 groups and treated as follows: saline as control (group 1), intraperitoneal cisplatin once a week for 3 weeks (group 2), and intraperitoneal cisplatin once a week for 3 weeks plus oral administration of d-methionine (group 3). Tissue somatic index (TSI), gastric emptying index (GEI), and feeding efficiency were measured. Both hepatic lipid metabolism and muscle atrophy-related gene expressions and C2C12 myotubes were determined by polymerase chain reaction. Micro-computed tomography (micro-CT) was used to conduct assessment of bone microarchitecture indices. Pathological changes of the gastric mucosa were assessed by hematoxylin and eosin staining after euthanizing the animals. d-Methionine increased food intake, weight gain, gastric emptying, and feeding efficiency, as well as decrease stomach contents, after cisplatin injections. Cisplatin caused shortening of myofibers. Cisplatin-induced muscle mass wasting was mediated by the elevation of mRNA expressions of MAFbx and MuRF-1 in ubiquitin ligases in muscle tissue homogenate. The mRNA expressions of MyoD and myogenin, markers of muscle differentiation, declined following cisplatin administration. The administration of d-methionine not only led to significant improvements in myofiber diameter and cross-sectional fiber areas but also reversed muscle atrophy-related gene expression. However, there were no significant changes in stomach histology or microarchitecture of trabecular bone among the study groups. The results indicate that d-methionine has an appetite-enhancing effect and ameliorates cisplatin-induced adipose and muscle tissue loss during cisplatin-based chemotherapy.

Chidamide-induced ROS accumulation and miR-129-3p-dependent cell cycle arrest in non-small lung cancer cells.

BACKGROUND: Epigenetic therapy is a promising popular treatment modality for various cancers. Histone modification and miRNA should not be underestimated in lung cancer. This study aimed to investigate whether chidamide, a histone deacetylase inhibitor (HDACi), which inhibits telomerase activity and induces cell cycle arrest, influences ROS and miRNA production in non-small cell lung cancer (NSCLC) cells. METHODS: H1355 and A549 were treated with chidamide. The analysis of DNA content was measured by FACSCalibur equipped with a 488 nm laser. H1355 cells were transfected with miR-129-3p mimic by Lipofectamine2000. Telomerase activity was performed on the telomeric repeat amplification protocol (TRAP) assay. Detection of thymidylate synthase (TS), p21, p53, pRB, and ß-actin, were performed by western blot analysis. RESULTS: Our data showed that expression of TS, p21, and pRB were altered in the presence of chidamide by PCR and western blot. Using BrdU-incorporation analysis, we found that chidamide induced G1 arrest through the regulation of the TS gene by miR-129-3p. Chidamide was shown to suppress telomerase activity in the TRAP assay and reduced the expression of human telomerase reverse transcriptase (hTERT) by PCR and q-PCR in H1355 and A549 cells. Chidamide increased the generation of reactive oxygen species (ROS) by flow cytometry. N-acetyl cysteine (NAC), a ROS scavenger, attenuated chidamide-induced telomerase activity inhibition. CONCLUSION: Chidamide repressed telomerase activity through ROS accumulation and cell cycle arrest by miR-129-3p upregulation in both H1355 and A549 cells. This is the first study to demonstrate that chidamide induces miR-129-3p upregulation and ROS accumulation, leading to cell cycle arrest.

Effects and mechanisms of betulinic acid on improving EGFR TKI-resistance of lung cancer cells.

Epidermal growth factor receptor (EGFR) mutations have been identified in approximately 55% of lung cancer patients in Taiwan. Gefitinib (Iressa) and Erlotinib (Tarceva) are the first-generation targeting drugs to patients with EGFR gene mutants a work by inhibiting tyrosine kinase activity. However, resistance in EGFR-mutated patients to first-generation tyrosine kinase inhibitor (TKI) therapy after 8-11 months of treatment has occurred. Betulinic acid (BetA) is a pentacyclic triterpenoid natural product derived from widespread plants. BetA has been reported to have a cytotoxic effect in several cancers. The purpose of this study is to investigate the effects and mechanisms of BetA on dampening EGFR TKI-resistance of lung cancer cells. Our study has demonstrated by MTT assay that combining BetA and an EGFR TKI increased the cytotoxicity against EGFR TKI-resistance lung cancer cells. Based on flow cytometry, combination treatments of BetA with an EGFR TKI enhanced Sub-G1 accumulation, induced apoptosis and induced mitochondrial membrane potential loss. Using western blotting, BetA and EGFR TKI combined treatments inhibited cell cycle related protein and triggered apoptosis- and autophagy- related protein expression. Taken together, our data suggests that a target therapy combining BetA with an EGFR TKI improves drug efficacy in EGFR TKI-resistant lung cancer cells.

Integrin ß3 and CD44 levels determine the effects of the OPN-a splicing variant on lung cancer cell growth.

Osteopontin (OPN), a phosphorylated glycoprotein, is frequently overexpressed in cancer. Among the three OPN isoforms, OPN-a is the most highly expressed in lung cancer cell lines and lung tumors. Overexpression of OPN-a greatly reduced CL1-5 lung adenocarcinoma cell growth, but had no effect on growth in A549 lung adenocarcinoma cells. Examination of the expression of integrins and CD44, which are possible OPN-a receptors, revealed that differences in integrin ß3 levels might explain this discrepancy between CL1-5 and A549 cells. When integrin ß3 was ectopically expressed in A549 cells, OPN-a inhibited their growth, whereas OPN-a increased cell growth following integrin ß3 knockdown in CL1-5 cells. This OPN-a-induced increase in growth appeared to result from activation of the CD44/NFκB pathway. Our results demonstrated that OPN-a inhibits growth of cells with high integrin ß3 levels and increases growth via activation of the CD44/NFκB pathway in cells with low integrin ß3 levels. Thus, OPN-a, integrin ß3, and CD44 interact to affect lung cancer cell growth, and this study may aid in the development of cancer treatment strategies involving these molecules.

The homeobox transcription factor Irxl1 negatively regulates MyoD expression and myoblast differentiation.

Irxl1/Mkx (Iroquois homeobox-like 1/Mohawk) encodes a member of the TALE subfamily of homeodomain proteins. It is expressed in multiple mesoderm-derived tissues and has recently been shown to regulate tendon differentiation during mouse embryonic development. Previously we showed that knockdown of Irxl1 in zebrafish caused a deficit in neural crest cells which consequently resulted in deformation of craniofacial muscles and arch cartilages. Here, we further demonstrate that loss of Irxl1 function results in deformed somites with disordered muscle fibers and myotendinous junctions. Because expression of myoD is increased in the somites of Irxl1 knockdown morphants, we test whether Irxl1 negatively regulates myoD expression. When stable C2C12 myoblasts overexpressing Irxl1/Mkx were induced to differentiate, myotube formation was inhibited and protein levels of myoD and myosin heavy chain were decreased accordingly. A series of deletion constructs of myoD promoter fragments were tested by luciferase reporter assays, which identified a promoter fragment that is necessary and sufficient for Irxl1-mediated repression. Direct interaction of Irxl1 and myoD promoter was subsequently elucidated by yeast one-hybrid assays, electrophoretic mobility shift assays and chromatin immunoprecipitation analysis. Furthermore, mouse Mkx also binds to and represses myoD promoter. These results indicate that Irxl1/Mkx can repress myoD expression through direct binding to its promoter and may thus play a negative regulatory role in muscle differentiation.

Surgical implantation of avulsed lumbosacral ventral roots promotes restoration of bladder morphology in rats.

Injuries to the cauda equina and conus medullaris of the spinal cord commonly result in paraplegia, sensory deficits, neuropathic pain, as well as bladder, bowel, and reproductive dysfunctions. In a recently developed lower motoneuron model for cauda equina injury and repair, we have demonstrated that an acute surgical implantation of avulsed lumbosacral ventral roots into the conus medullaris is neuroprotective, promotes regeneration of efferent spinal cord axons into the implanted roots, and may result in functional reinnervation of the lower urinary tract. Here, we investigated the effects of a bilateral lumbosacral ventral root avulsion (VRA) injury and re-implantation on the morphology of the rat bladder at twelve weeks post-operatively. We demonstrated a VRA-induced overall thinning of the bladder wall, which exhibited reduced thickness of both the lamina propria and smooth muscle. In contrast, the bladder epithelium markedly increased its thickness in the injured series. Quantitative immunohistochemical studies showed a selective increase in CGRP immunoreactivity in the lamina propria after the VRA injury. Interestingly, the injury-induced changes in bladder wall morphology were ameliorated by an acute implantation of the lesioned roots into the conus medullaris. Specifically, bladders of the implanted group showed a partial restoration of the thickness of the lamina propria and epithelium as well as a return of CGRP immunoreactivity to baseline levels in the lamina propria. Our results support the notion that surgical implantation of severed ventral roots into the spinal cord may promote the recovery of a normal morphological phenotype in peripheral end organs.

Differential effects of urethane and isoflurane on external urethral sphincter electromyography and cystometry in rats.

Urethane is a common and often preferred anesthetic agent for urodynamic recordings in rats, but its use is often restricted to terminal procedures because of a prolonged duration of action and potentially toxic effects. When urodynamic recordings are part of survival procedures in rodent experimental models, inhalation anesthetics, such as isoflurane, are frequently used and generally well tolerated. In this study, we compared the effects of urethane and isoflurane on lower urinary tract function. For this purpose, adult female rats were anesthetized by subcutaneous administration of urethane (n=6) or by inhalation of isoflurane (n=5). Micturition reflexes were assessed by concurrent cystometrogram and external urethral sphincter (EUS) electromyography (EMG) recordings to determine bladder contractile properties, EUS activation patterns, and the coordination between bladder contractions and EUS activation. Compared with urethane, isoflurane reduced frequency of bursts, firing frequency, and amplitude of EUS EMG activity during voiding as well as the EUS EMG amplitude during the bladder filling phase. Isoflurane also prolonged the bladder intercontractile intervals. Other several key functional aspects of the bladder contractile properties as well as the coordination between bladder contractions were not different between the two experimental groups. We conclude that micturition reflexes were differentially affected by isoflurane and urethane. Specifically, isoflurane exhibited a significant suppression of the EUS EMG activity and prolonged the bladder intercontractile intervals compared with urethane. We suggest that these anesthetic properties be taken into consideration during the experimental design and interpretation of urodynamic recordings in rodent models.

Re-established micturition reflexes show differential activation patterns after lumbosacral ventral root avulsion injury and repair in rats.

Previous studies have demonstrated that an acute implantation of lesioned lumbosacral ventral roots into the rat conus medullaris (CM) results in functional reinnervation of the lower urinary tract (LUT). Although the root implantation procedure results in a return of reflexive micturition, voiding efficiency (VE) remains incompletely recovered. Here, we performed a detailed urodynamic analysis of cystometry and external urethral sphincter (EUS) electromyography (EMG) recordings to determine underlying mechanisms for the incompletely recovered VE. For this purpose, adult female rats were studied at 12 weeks after a bilateral L5-S2 ventral root avulsion injury followed by an acute surgical implantation of the avulsed L6 and S1 ventral roots into the CM (n=6). Age-matched sham-operated rats (n=6) were included for control purposes. Compared to sham-operated controls, rats of the implanted series showed 1) reflex bladder contractions with a significantly shortened urine expulsion phase, 2) markedly decreased phasic EUS EMG activity during micturition, and 3) a pronounced bladder-sphincter dys-coordination, as demonstrated by a significantly delayed onset of the switch from low-amplitude tonic EUS EMG activity to either phasic EUS EMG activity or a large-amplitude tonic EUS EMG activity during the urine expulsion phase. Our findings provide a mechanistic explanation for the incomplete recovery of the VE following implantation of avulsed ventral roots into the spinal cord. Our future studies will aim to increase successful axonal regeneration in attempts to augment the recovery of the LUT after cauda equina injury and repair.

Serotonergic drugs and spinal cord transections indicate that different spinal circuits are involved in external urethral sphincter activity in rats.

Lower urinary tract function is regulated by spinal and supraspinal reflexes that coordinate the activity of the urinary bladder and external urethral sphincter (EUS). Two types of EUS activity (tonic and bursting) have been identified in rats. This study in urethane-anesthetized female rats used cystometry, EUS electromyography, spinal cord transection (SCT) at different segmental levels, and analysis of the effects of 5-HT(1A) receptor agonist (8-OH-DPAT) and antagonist (WAY100635) drugs to examine the origin of tonic and bursting EUS activity. EUS activity was elicited by bladder distension or electrical stimulation of afferent axons in the pelvic nerve (pelvic-EUS reflex). Tonic activity evoked by bladder distension was detected in spinal cord-intact rats and after acute and chronic T8-9 or L3-4 SCT but was abolished after L6-S1 SCT. Bursting activity was abolished by all types of SCT except chronic T8-9 transection. 8-OH-DPAT enhanced tonic activity, and WAY100635 reversed the effect of 8-OH-DPAT. The pelvic-EUS reflex consisted of an early response (ER) and late response (LR) when the bladder was distended in spinal cord-intact rats. ER remained after acute or chronic T8-9 and L3-4 SCT, but was absent after L6-S1 SCT. LR occurred only in chronic T8-9 SCT rats where it was enhanced or unmasked by 8-OH-DPAT. The results indicate that spinal serotonergic mechanisms facilitate tonic and bursting EUS activity. The circuitry for generating different patterns of EUS activity appears to be located in different segments of the spinal cord: tonic activity at L6-S1 and bursting activity between T8-9 and L3-4.

External urethral sphincter activity in a rat model of pudendal nerve injury.

AIMS: Pudendal nerve injury in the rat has been a useful animal model for studying stress urinary incontinence (SUI). However, the effect of pudendal nerve injury on activity of the external urethral sphincter (EUS) is relatively unexplored. The aims of this study were to examine voiding and the EUS electromyogram (EMG) in a durable SUI model in rats with bilateral or unilateral pudendal nerve transections. In addition, the effects of denervation on urethral anatomy were investigated. METHODS: A leak point pressure (LPP) test was first used to demonstrate that pudendal nerve transection induced SUI. Cystometry exhibited changes in voiding function and EUS-EMG measurements provided a quantitative evaluation of EUS activity during voiding. The morphological changes in sections through the mid-urethra were assessed with hematoxylin and eosin (H&E) staining. RESULTS: A significant decrease in average LPP was detected in rats 6 weeks after bilateral pudendal nerve transection (BPNT). Abnormal urodynamic measurements including a decrease in contraction amplitude and voided volume as well as an increase in contraction duration, and residual volume all indicated inefficient voiding. In addition EUS-EMG silent periods were reduced and the frequency of EUS-EMG bursting during voiding was increased. Atrophy of striated muscle in the EUS was also detected in rats with pudendal nerve transection(s). CONCLUSIONS: Our results indicate that pudendal nerve transection in rats decreases urethral outlet resistance and causes striated muscle atrophy in the EUS, EUS-EMG abnormalities and inefficient voiding. The results demonstrate that BPNT is a durable model for SUI.

Roles of glutamatergic and serotonergic mechanisms in reflex control of the external urethral sphincter in urethane-anesthetized female rats.

This study was conducted to examine reflex mechanisms that mediate urinary bladder and external urethral sphincter (EUS) coordination in urethane-anesthetized female Sprague-Dawley rats. We investigated the properties of EUS reflexes elicited by electrical stimulation of pelvic nerve afferent axons (pelvic-EUS reflex). The changes in the reflexes induced by bladder distension and administration of agonists or antagonists for glutamatergic or serotonergic receptors were examined. The reflexes consisted of an early response (ER, 18- to 22-ms latency) and a late, long-duration (>100-ms latency) response (LR), which consisted of bursts of activity at 20- to 160-ms interburst intervals. In a few experiments, a reflex with an intermediate (40- to 70-ms) latency was also identified. With the bladder empty, the ER, but not the LR, was detected in the majority of experiments. The LR was markedly enhanced when the bladder was distended. The ER remained, but the LR was abolished, after spinal cord transection at T8-T9. The ER and LR were significantly decreased 75 and 35%, respectively, by the N-methyl-D-aspartate receptor antagonist MK-801 (0.3 mg/kg iv), but only decreased 18 and 14%, respectively, by the alpha-amino-5-methylisoxazole-4-propionate receptor antagonist LY-215490 (3 mg/kg iv). The serotonin (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (1 mg/kg iv) enhanced spontaneous EUS activity and the pelvic-EUS reflex. WAY-100635 (0.1-1 mg/kg iv), a 5-HT1A antagonist, reversed the effect of 8-hydroxy-2-(di-n-propylamino)-tetralin and suppressed EUS activity and the pelvic-EUS reflex. These results indicate that glutamatergic and serotonergic mechanisms are important in the reflex pathways underlying bladder- sphincter coordination in rats.

Reflexes evoked by electrical stimulation of afferent axons in the pudendal nerve under empty and distended bladder conditions in urethane-anesthetized rats.

This study examined reflex mechanisms that mediate urinary bladder and external urethral sphincter (EUS) coordination in female Sprague-Dawley urethane-anesthetized rats under empty and distended bladder conditions. The bladder was distended either by a small balloon or a saline filled catheter inserted through the body of the bladder. Stimulation of the entire pudendal nerve elicited short latency (8-12 ms) responses in the EUS and short (3-8 ms) and long latency responses (16-20 ms) in contralateral pudendal nerve. The long latency pudendal-pudendal reflex was reduced by 36.7% in area during bladder distension with the balloon catheter. However, there was no significant change in the area of pudendal-EUS reflex during bladder distension. Peak amplitudes of both reflexes were reduced 32% by bladder distension. The effects of glutamatergic receptor antagonists on the reflexes were also examined. MK 801 (0.3-5mg/kg, i.v.), an N-methyl-d-aspartate glutamatergic receptor antagonist, markedly depressed the pudendal-pudendal reflex, but LY 215490 (3mg/kg, i.v.), an alpha-amino-5-methyl isoxazole-4-propionate antagonist, had a minimal inhibitory effect. Both glutamatergic receptor antagonists significantly suppressed the pudendal-EUS reflex. These results indicate that the EUS is innervated by multiple pathways and that glutamatergic excitatory transmission is important in the neural mechanisms underlying bladder-sphincter coordination in the rat.