Rehabilitation Program for Postlaryngectomy Patients Following Ileocolon Flap Transfer for Voice Reconstruction: An Essential Part of Success.

BACKGROUND: Speech restoration is important for communication and social activities after pharyngolaryngectomy in head and neck cancer or corrosive injury. Several techniques of voice restoration have been developed to improve life quality. The aim of this paper was to focus on the microsurgical transfer of ileocolon flap and outcome of further voice rehabilitation. PATIENTS AND METHODS: From 2010 to 2022, 69 patients had ileocolon flap at our hospital with postoperative speech training and regular follow-up for over 1 year. The patients received deglutition training first, followed by voice rehabilitation. Voice outcomes were evaluated at an interval of 3 months and finally at 12 months of voice training rehabilitation. Among other examinations, the speech function was evaluated using a 4-point Likert scale and senior surgeon (H-c.C.) scoring system. RESULTS: The results showed that speech function reached 13.1% of excellent voice, 65.1% of good voice, 13.1% of fair result, and 8.7% of poor result by Likert scales. Meanwhile, the senior surgeon (H-c.C.) score showed 17.4% of excellent, 63.8% of moderate, and 18.8% of poor results. About voice laboratory results, maximal phonation time was 11.0 seconds, and the average number counted in one breath was 15. Loudness and frequency showed 56.0 dB and 105.0 Hz, respectively. CONCLUSION: The study showed that after voice reconstruction with ileocolon flap followed by the voice rehabilitation program, the patients would have a better understanding of the altered anatomical structures and practice in a more efficient way. Adequate recommendation by the therapists to plastic surgeons for revision surgeries optimized voice function of the patients.

Coenzyme Q0 defeats NLRP3-mediated inflammation, EMT/metastasis, and Warburg effects by inhibiting HIF-1α expression in human triple-negative breast cancer cells.

Coenzyme Q0 (CoQ0) is a derivative quinone from Antrodia camphorata (AC) that exerts anticancer activities. This study examined the anticancer attributes of CoQ0 (0-4 µM) on inhibited anti-EMT/metastasis and NLRP3 inflammasome, and altered Warburg effects via HIF-1α inhibition in triple-negative breast cancer (MDA-MB-231 and 468) cells. MTT assay, cell migration/invasion assays, Western blotting, immunofluorescence, metabolic reprogramming, and LC-ESI-MS were carried out to assess the therapy potential of CoQ0. CoQ0 inhibited HIF-1α expression and suppressed the NLRP3 inflammasome and ASC/caspase-1 expression, followed by downregulation of IL-1ß and IL-18 expression in MDA-MB-231 and 468 cells. CoQ0 ameliorated cancer stem-like markers by decreasing CD44 and increasing CD24 expression. Notably, CoQ0 modulated EMT by upregulating the epithelial marker E-cadherin and downregulating the mesenchymal marker N-cadherin. CoQ0 inhibited glucose uptake and lactate accumulation. CoQ0 also inhibited HIF-1α downstream genes involved in glycolysis, such as HK-2, LDH-A, PDK-1, and PKM-2 enzymes. CoQ0 decreased extracellular acidification rate (ECAR), glycolysis, glycolytic capacity, and glycolytic reserve in MDA-MB-231 and 468 cells under normoxic and hypoxic (CoCl2) conditions. CoQ0 inhibited the glycolytic intermediates lactate, FBP, and 2/3-PG, and PEP levels. CoQ0 increased oxygen consumption rate (OCR), basal respiration, ATP production, maximal respiration, and spare capacity under normoxic and hypoxic (CoCl2) conditions. CoQ0 increased TCA cycle metabolites, such as citrate, isocitrate, and succinate. CoQ0 inhibited aerobic glycolysis and enhanced mitochondrial oxidative phosphorylation in TNBC cells. Under hypoxic conditions, CoQ0 also mitigated HIF-1α, GLUT1, glycolytic-related (HK-2, LDH-A, and PFK-1), and metastasis-related (E-cadherin, N-cadherin, and MMP-9) protein or mRNA expression in MDA-MB-231 and/or 468 cells. Under LPS/ATP stimulation, CoQ0 inhibited NLRP3 inflammasome/procaspase-1/IL-18 activation and NFκB/iNOS expression. CoQ0 also hindered LPS/ATP-stimulated tumor migration and downregulated LPS/ATP-stimulated N-cadherin and MMP-2/-9 expression. The present study revealed that suppression of HIF-1α expression caused by CoQ0 may contribute to inhibition of NLRP3-mediated inflammation, EMT/metastasis, and Warburg effects of triple-negative breast cancers.

Antrodia camphorata and coenzyme Q0 , a novel quinone derivative of Antrodia camphorata, impede HIF-1α and epithelial-mesenchymal transition/metastasis in human glioblastoma cells.

Antrodia camphorata (AC) and Coenzyme Q0 (CoQ0 ), a novel quinone derivative of AC, exhibits antitumor activities. The present study evaluated EMT/metastasis inhibition and autophagy induction aspects of AC and CoQ0 in human glioblastoma (GBM8401) cells. Our findings revealed that AC treatment (0-150 µg/mL) hindered tumor cell proliferation and migration/invasion in GBM8401 cells. Notably, AC treatment inhibited HIF-1α and EMT by upregulating epithelial marker protein E-cadherin while downregulating mesenchymal proteins Twist, Slug, Snail, and ß-catenin. There was an appearance of the autophagy markers LC3-II and p62/SQSTM1, while ATG4B was downregulated by AC treatment. We also found that CoQ0 (0-10 µM) could inhibit migration and invasion in GBM8401 cells. In particular, E-cadherin was elevated and N-cadherin, Vimentin, Twist, Slug, and Snail, were reduced upon CoQ0 treatment. In addition, MMP-2/-9 expression and Wnt/ß-catenin pathways were downregulated. Furthermore, autophagy inhibitors 3-MA or CQ reversed the CoQ0 -elicited suppression of migration/invasion and metastasis-related proteins (Vimentin, Snail, and ß-catenin). Results suggested autophagy-mediated antiEMT and antimetastasis upon CoQ0 treatment. CoQ0 inhibited HIF-1α and metastasis in GBM8401 cells under normoxia and hypoxia. HIF-1α knockdown using siRNA accelerated CoQ0 -inhibited migration. Finally, CoQ0 exhibited a prolonged survival rate in GBM8401-xenografted mice. Treatment with Antrodia camphorata/CoQ0 inhibited HIF-1α and EMT/metastasis in glioblastoma.

Anticancer activities of chalcone flavokawain B from Alpinia pricei Hayata in human lung adenocarcinoma (A549) cells via induction of reactive oxygen species-mediated apoptotic and autophagic cell death.

Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non-small-cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0-15 µg/ml) decreases cell viability and colony formation, dysregulates the Bax:B-cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase-9/-3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB-induced apoptosis. In addition, FKB-induced reactive oxygen species (ROS) generation, and N-acetylcysteine attenuated FKB-induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule-related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS-mediated ATG4B expression. Inhibiting autophagy using 3-methyladenine/chloroquine diminished FKB-induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS-mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.

Ubiquitin-specific protease 3 overexpression promotes gastric carcinogenesis and is predictive of poor patient prognosis.

Although gastric cancer (GC) is one of the most common cancers, knowledge of its development and carcinogenesis is limited. To date, expression of ubiquitin-specific protease 3 (USP3) in all types of cancer, including GC, is still unknown. The present study explored the involvement of USP3 in the carcinogenesis and prognosis of GC. We measured USP3 expression in normal and GC tissues and cell lines. Correlations between USP3 protein level and clinicopathological parameters, as well as the significance of USP3 protein level for disease-free survival were assessed. Small hairpin RNA technology and transfection were used to investigate the effect of USP3 manipulation on cell proliferation and spreading. Moreover, xenograft proliferation and metastasis were used to explore the influence of USP3 on tumor growth and metastasis in animals. An increase in USP3 expression was observed in GC cells and tissues. The overexpression of USP3 was significantly correlated with several clinicopathological parameters and poor disease-free survival. Multivariate Cox regression analysis showed that the overexpression of USP3 was an independent prognostic biomarker. Silencing of USP3 suppressed GC cell proliferation and spreading in vitro as well as xenograft proliferation and metastasis in vivo; however, opposite results were obtained when USP3 was overexpressed. Further studies showed that USP3 influenced cell proliferation and spreading by regulating the cell cycle control- and epithelial-mesenchymal transition-related molecules. This study suggests that USP3 overexpression can be a useful biomarker for predicting the outcomes of GC patients and that USP3 targeting represents a potential modality for treating GC.

Ceramide synthase 6 predicts the prognosis of human gastric cancer: It functions as an oncoprotein by dysregulating the SOCS2/JAK2/STAT3 pathway.

Although gastric cancer (GC) is one of the most common cancers, knowledge of its development, and carcinogenesis is limited. The present study explored the involvement of ceramide synthase 6 (CERS6) in GC carcinogenesis and prognosis. RT-PCR, immunoblotting, and immunohistochemistry were used to examine the expression of CERS6. Transfection and small hairpin RNA technology were used to investigate the effect of CERS6 manipulation on cell proliferation and spread as well as the underlying mechanism. Moreover, xenograft proliferation was employed to explore the influence of CERS6 on tumor growth in animals. It was found that overexpression of CERS6 was significantly correlated with several clinicopathologic parameters and poor disease-free survival. The overexpression and silencing of CERS6 in GC cells facilitated and suppressed cell proliferation and spread as well as xenograft proliferation, respectively. Mechanistic studies further revealed that CERS6 influenced cell proliferation and spread by regulating cell cycle control and metastasis-related protein through the SOCS2/JAK2/STAT3 signaling pathway. Collectively, this study suggests that CERS6 overexpression could be a useful biomarker for predicting the outcomes of GC patients and that CERS6 targeting represents a potential modality for treating GC.

Application of transoral continuous intraoperative neuromonitoring in natural orifice transluminal endoscopic surgery for thyroid disease: a preliminary study.

BACKGROUND: The novel concept of continuous intraoperative neuromonitoring (Cont-IONM) through stimulation of the vagal nerve has been used in thyroidectomies to prevent imminent injury of the recurrent laryngeal nerve (RLN). This article reports on this technology and the results of using transoral Cont-IONM in natural orifice transluminal endoscopic surgery for thyroid disease. METHODS: Cont-IONM of the RLN was achieved through automatic cyclical stimulation of the vagal nerve using a C2 monitor and delta stimulating electrode. During the operation, three vestibular incisions were made, and the stimulating electrode was transorally inserted, with its cable line lying outside the trocar. The vagal nerve was gently dissected, looped, and then enveloped by the electrode cuff. Electromyography (EMG) of the vocalis muscle was performed, and the alarm was set to activate when the EMG amplitude reduced by 50% and latency was prolonged by 10%. Demographic data and outcome variables, including incremental time required to achieve Cont-IONM, were obtained. RESULTS: A total of 20 patients (28 nerves at risk) undergoing a transoral endoscopic thyroidectomy vestibular approach were enrolled in this study. All Cont-IONM procedures were successfully completed. In all patients, the stimulation was set at 0.7 milliamps every 1 s, and Cont-IONM use was unassociated with any untoward neural, cardiovascular, or gastrointestinal sequelae. On average, the ipsilateral Cont-IONM procedure required 10.33 ± 2.57 min to complete. Except for one instance, no significant problems occurred with electrode displacement. In one patient, a combined EMG event occurred, which improved after releasing the thyroid retractor, and the patient had no vocal cord paralysis postoperatively. CONCLUSION: Cont-IONM is feasible and safe to use during transoral endoscopic thyroidectomies and may assist in the early detection of adverse EMG changes, thereby preventing paralysis of the RLNs.

Chalcone flavokawain B induces autophagic-cell death via reactive oxygen species-mediated signaling pathways in human gastric carcinoma and suppresses tumor growth in nude mice.

Flavokawain B (FKB), a naturally occurring chalcone in kava extracts, has been reported to possess anticancer activity. However, the effect of FKB on gastric cancer remains unclear. We examined the in vitro and in vivo anticancer activity and autophagy involvement of FKB and determined the underlying molecular mechanisms. FKB is potently cytotoxic to human gastric cancer cells (AGS/NCI-N87/KATO-III/TSGH9201) and mildly toxic towards normal (Hs738) cells and primary mouse hepatocytes. FKB-induced AGS cell death was characterized by autophagy, not apoptosis, as evidenced by increased LC3-II accumulation, GFP-LC3 puncta and acidic vesicular organelles (AVOs) formation, without resulting procaspase-3/PARP cleavage. FKB further caused p62/SQSTM1 activation, mTOR downregulation, ATG4B inhibition, and Beclin-1/Bcl-2 dysregulation. Silencing autophagy inhibitors CQ/3-MA and LC3 (shRNA) significantly reversed the FKB-induced cell death of AGS cells. FKB-triggered ROS generation and ROS inhibition by NAC pre-treatment diminished FKB-induced cell death, LC3 conversion, AVO formation, p62/SQSTM1 activation, ATG4B inhibition and Beclin-1/Bcl-2 dysregulation, which indicated ROS-mediated autophagy in AGS cells. Furthermore, FKB induces G2/M arrest and alters cell-cycle proteins through ROS-JNK signaling. Interestingly, FKB-induced autophagy is associated with the suppression of HER-2 and PI3K/AKT/mTOR signaling cascades. FKB inhibits apoptotic Bax expression, and Bax-transfected AGS cells exhibit both apoptosis and autophagy; thus, FKB-inactivated Bax results in apoptosis inhibition. In vivo data demonstrated that FKB effectively inhibited tumor growth, prolonged the survival rate, and induced autophagy in AGS-xenografted mice. Notably, silencing of LC3 attenuated FKB-induced autophagy in AGS-xenografted tumors. FKB may be a potential chemopreventive agent in the activation of ROS-mediated autophagy of gastric cancer cells.

High expression of mitochondrial intermembrane chaperone TIMM9 represents a negative prognostic marker in gastric cancer.

BACKGROUND/PURPOSE: Gastric cancer (GC) is one of the most common malignant cancers worldwide. However, little is known about the molecular process underlying this disease and its progression. This study investigated correlations between the expression of a mitochondrial inner membrane protein translocase of inner mitochondrial membrane 9 homolog (TIMM9) and various clinicopathologic parameters as well as patients' survival. METHODS: Gastric tissue samples were obtained from 140 patients with GC and expression levels of TIMM9 were analyzed through immunohistochemistry. Paired t tests were used to analyze the differences in the expression levels of TIMM9 in both tumor and nontumor tissues for each patient. Two-tailed χ2 tests were performed to determine whether the differences in TIMM9 expression and clinicopathologic parameters were significant. Time-to-event endpoints for clinicopathologic parameters were plotted using the Kaplan-Meier method, and statistical significance was determined using univariate log-rank tests. Cox proportional hazard model was used for multivariate analysis to determine the independence of prognostic effects of TIMM9 expression. RESULTS: A borderline association was found between overexpression of TIMM9 and vascular invasion (p = 0.0887). Patients with high expression levels of TIMM9 achieved a significantly lower disease-free survival rate compared with those with low expression levels (p = 0.005). Multivariate Cox regression analysis showed that overexpression of TIMM9 was an independent prognostic marker for GC (p = 0.011). CONCLUSION: Overexpression of TIMM9 can be used as a marker to predict the outcome of patients with GC.

Frequent DNA methylation of MiR-129-2 and its potential clinical implication in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with poor prognosis and high mortality due to a lack of effective medical treatment and apparent early stage symptoms. Understanding molecular mechanism of cancer development is crucial for HCC diagnosis, prognosis, and treatment. Recently, microRNAs have been shown to play an important role in carcinogenesis, being regulated by DNA methylation in several cases. In this study, a whole genome approach was used to identify methylation-regulated miRNAs in HCC, finally focusing on miR-129-2. MiR-129-2 methylation and reduced expression were observed in all examined HCC cell lines but not in normal liver cells and tissues. In 39 (93%) of 42 HCC, the methylation levels of miR-129-2 were significantly increased in tumor tissues compared with adjacent normal tissues. Furthermore, miR-129-2 methylation was detectable in plasma samples from HCC patients, but not in plasma samples from healthy individuals or patients with liver cirrhosis. At a cut-off value of -2.36 (log2 transformation of methylation level), it was possible to distinguish HCC from healthy and cirrhotic controls with sensitivity and specificity of 88% and 100%, respectively. This study indicates that miR-129-2 methylation is highly accurate in distinguishing HCC patients from cirrhosis patients and healthy individuals, implying its potential utility as an early diagnostic marker for HCC.

Punicalagin Restricts Growth, Promotes Apoptosis, and Reduces Invasion in Human Gastric Cancer Cells.

This research investigated the anticancer properties of punicalagin, a prominent bioactive polyphenol extracted from Punica granatum L, in human gastric cancer cell lines. Normal and gastric cancer cells were exposed to different doses of punicalagin for various durations. Punicalagin exhibited cytotoxic effects on gastric cancer cells in a dose- and time-dependent fashion, while sparing normal gastric epithelial cells. It is noteworthy that among the 3 gastric cancer cells, HGC-27 cells were more resistant to punicalagin than 23,132/87 and AGS cells. Furthermore, punicalagin triggered apoptosis in gastric cancer cells, evidenced by a rise in both early and late apoptotic cell percentages. Western blot analysis further revealed that punicalagin elevated the levels of activated caspase-3. Conversely, punicalagin curtailed cell invasion and reduced the expression of MMP-2, MMP-9, Snail, and Slug. From a mechanistic standpoint, Western blotting indicated that punicalagin might inhibit the Erk and NF-κB pathways, leading to apoptosis induction and the inhibition of cell invasion in gastric cancer cells. These results indicate that punicalagin promotes apoptosis and inhibits cell invasion in gastric cancer cells by activating caspase-3 and suppressing MMP-2, MMP-9, Snail, and Slug through the inhibition of the Erk and NF-κB pathways.

Punicalagin is cytotoxic to human colon cancer cells by modulating cell proliferation, apoptosis, and invasion.

Purpose: The purpose of this study was to explore the anticancer effect of punicalagin, an abundant bioactive tannin compound isolated from Punica granatum L., on three colon cancer cell lines, namely, HCT 116, HT-29, and LoVo.Research Design: Normal and colon cancer cells were treated with different concentrations of punicalagin for different periods. Data Collection and Analysis: Cell viability was measured with a CCK-8 assay. Programmed cell death and invasion were analyzed using an annexin V and cell death kit and a cell invasion analysis kit. The expression of active caspase-3, MMP-2, MMP-9, Snail, and Slug were measured by Western blot.Results: The results of the cell viability analysis showed that punicalagin was cytotoxic to colon cancer cells, but it was not to normal cells in a dose- and time-dependent manner. Additionally, punicalagin induced apoptosis in colon cancer cells (shown by the cumulative percentage of colorectal cancer cells in early and late apoptosis). It was found that caspase-3 activity increased following punicalagin treatment. Western blot results also showed that punicalagin increased the expression of activated caspase-3. In contrast, punicalagin inhibited the invasion of colon cancer cells. Further, treatment of colon cancer cells with punicalagin suppressed the expression of MMP-2, MMP-9, Snail, and Slug. Conclusions: These results showed that the activation of caspase-3 and the inhibition of MMP-2, MMP-9, Snail and Slug were involved in the effects of punicalagin on colon cancer cells.

NCAPG deregulation indicates poor patient survival and contributes to colorectal carcinogenesis.

Colorectal cancer (CRC) is one of the types of cancers with a high incidence and is ranked the 3rd among men and 2nd among women worldwide. The purpose of this study was to investigate the correlation between non-SMC condensin I complex subunit G (NCAPG) and the prognosis of CRC and its function in CRC cells. The expression of NCAPG in colorectal tissues and cells was detected by immunoblotting and immunohistochemistry. Kaplan-Meier analysis was used to analyze the correlation between NCAPG and CRC prognosis. RNAi technology was used to investigate how NCAPG inhibition affected the proliferation and migration of CRC cells. Overexpression of NCAPG was positively correlated with several clinicopathologic characteristics, including T stage (P = 0.0198), M stage (P = 0.0005), and TNM stage (P < 0.0001). Kaplan-Meier analysis showed that the overexpression of NCAPG was also negatively correlated with disease-free survival and overall survival. In the culture of CRC cells, the knockdown of NCAPG inhibited the proliferation, migration, and invasion of the cells. Meanwhile, it was also found that NCAPG knockdown could interfere with G2/M-G1 transition in the cell cycle, resulting in the inhibition of cell proliferation. The overexpression of NCAPG may serve as a candidate biomarker for CRC prognosis. NCAPG is also a potential therapeutic target for CRC.

The in vitro and in vivo anticancer activities of Antrodia salmonea through inhibition of metastasis and induction of ROS-mediated apoptotic and autophagic cell death in human glioblastoma cells.

BACKGROUND: Antrodia salmonea (AS) exhibits anticancer activities against various cancers. OBJECTIVE: This study investigated the anticancer activities of AS on human glioblastoma (GBM8401 and U87MG) cells both in vitro and in vivo and explained the underlying molecular mechanism. METHODS: MTT, colony formation, migration/invasion assay, immunoblotting, immunofluorescence, TUNEL, Annexin V/PI staining, AO staining, GFP-LC3 transfection, TEM, qPCR, siLC3, DCFH2-DA assay, and xenografted-nude mice were used to assess the potential of AS therapy. RESULTS: AS treatment retarded growth and suppressed colony formation in glioblastoma cells. AS attenuates EMT by suppressing invasion and migration, increasing E-cadherin expression, decreasing Twist, Snail, and N-cadherin expression, and inhibiting Wnt/ß-catenin pathways in GBM8401 and U87MG cells. Furthermore, AS induced apoptosis by activating caspase-3, cleaving PARP, and dysregulating Bax and Bcl-2 in both cell lines. TUNEL assay and Annexin V/PI staining indicated AS-mediated late apoptosis. Interestingly, AS induced autophagic cell death by LC3-II accumulation, AVO formation, autophagosome GFP-LC3 puncta, p62/SQSTM1 expression, and ATG4B inhibition in GBM8401 and U87MG cells. TEM data revealed that AS favored autophagosome and autolysosome formation. The autophagy inhibitors 3-MA/CQ and LC3 knockdown suppressed AS-induced apoptosis in glioblastoma cells, indicating that the inhibition of autophagy decreased AS-induced apoptosis. Notably, the antioxidant N-acetylcysteine (NAC) inhibited AS-mediated ROS production and AS-induced apoptotic and autophagic cell death. Furthermore, AS induced ROS-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. AS reduced the tumor burden in GBM8401-xenografted nude mice and significantly modulated tumor xenografts by inducing anti-EMT, apoptosis, and autophagy. AS could be a potential antitumor agent in human glioblastoma treatment.

The Antitumor Activity of Antrodia camphorata in Melanoma Cells: Modulation of Wnt/ß-Catenin Signaling Pathways.

Antrodia camphorata (AC) is well known in Taiwan as a traditional Chinese medicine. The aim of this study was to investigate whether a fermented culture broth of AC could inhibit melanoma proliferation and progression via suppression of the Wnt/ß-catenin signaling pathway. In this study, we observed that AC treatment resulted in decreased cell viability and disturbed Wnt/ß-catenin cascade in B16F10 and/or B16F1 melanoma cells. This result was accompanied by a decrease in the expression of Wnt/ß-catenin transcriptional targets, including c-Myc and survivin. Furthermore, treatment of melanoma cells with AC resulted in a significant increase in apoptosis, which was associated with DNA fragmentation, cytochrome c release, caspase-9 and -3 activation, PARP degradation, Bcl-2/Bax dysregulation, and p53 expression. We also observed that AC caused G(1) phase arrest mediated by a downregulation of cyclin D1 and CDK4 and increased p21 and p27 expression. In addition, we demonstrated that non- and subcytotoxic concentrations of AC markedly inhibited migration and invasion of highly metastatic B16F10 cells. The antimetastatic effect of AC was further confirmed by reductions in the levels of MMP-2, MMP-9, and VEGF expression. These results suggest that Antrodia camphorata may exert antitumor activity by downregulating the Wnt/ß-catenin pathways.

Inhibition of Cell Growth and Induction of Apoptosis by Antrodia camphorata in HER-2/neu-Overexpressing Breast Cancer Cells through the Induction of ROS, Depletion of HER-2/neu, and Disruption of the PI3K/Akt Signaling Pathway.

Previously, we demonstrated that a submerged fermentation culture of Antrodia camphorata (AC) promotes cell-cycle arrest and apoptosis in human estrogen receptor-positive/negative breast cancer cells. However, whether AC is effective against HER-2/neu-overexpressing breast cancers has not been thoroughly elucidated. In the present study, we showed that AC exhibited a significant cytotoxic effect against HER-2/neu-overexpressing MDA-MB-453 and BT-474 cells. Immunoblot analysis demonstrated that HER-2/neu and their tyrosine phosphorylation were inhibited by AC in a dose-dependent manner. An increase in intracellular reactive oxygen species (ROS) was observed in AC-treated cells, whereas antioxidant N-acetylcysteine (NAC) significantly prevented AC induced HER-2/neu depletion and cell death, which directly indicates that AC-induced HER-2/neu depletion and cell death was mediated by ROS generation. Also, AC significantly downregulated the expression of cyclin D1, cyclin E, and CDK4 followed by the suppression of PI3K/Akt, and their downstream effectors GSK-3ß and ß-catenin. Notably, AC-treatment induced apoptotic cell death, which was associated with sub-G1 accumulation, DNA fragmentation, mitochondrial dysfunction, cytochrome c release, caspase-3/-9 activation, PARP degradation, and Bcl-2/Bax dysregulation. Assays for colony formation also confirmed the growth-inhibitory effects of AC. This is the first report confirming the anticancer activity of this potentially beneficial mushroom against human HER-2/neu-overexpressing breast cancers.

Coenzyme Q0 Inhibits NLRP3 Inflammasome Activation through Mitophagy Induction in LPS/ATP-Stimulated Macrophages.

Coenzyme Q (CoQ) analogs with a variable number of isoprenoid units have exhibited as anti-inflammatory as well as antioxidant molecules. Using novel quinone derivative CoQ0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone, zero side chain isoprenoid), we studied its molecular activities against LPS/ATP-induced inflammation and redox imbalance in murine RAW264.7 macrophages. CoQ0's non- or subcytotoxic concentration suppressed the NLRP3 inflammasome and procaspase-1 activation, followed by downregulation of IL1ß expression in LPS/ATP-stimulated RAW264.7 macrophages. Similarly, treatment of CoQ0 led to LC3-I/II accumulation and p62/SQSTM1 activation. An increase in the Beclin-1/Bcl-2 ratio and a decrease in the expression of phosphorylated PI3K/AKT, p70 S6 kinase, and mTOR showed that autophagy was activated. Besides, CoQ0 increased Parkin protein to recruit damaged mitochondria and induced mitophagy in LPS/ATP-stimulated RAW264.7 macrophages. CoQ0 inhibited LPS/ATP-stimulated ROS generation in RAW264.7 macrophages. Notably, when LPS/ATP-stimulated RAW264.7 macrophages were treated with CoQ0, Mito-TEMPO (a mitochondrial ROS inhibitor), or N-acetylcysteine (NAC, a ROS inhibitor), there was a significant reduction of LPS/ATP-stimulated NLRP3 inflammasome activation and IL1ß expression. Interestingly, treatment with CoQ0 or Mito-TEMPO, but not NAC, significantly increased LPS/ATP-induced LC3-II accumulation indicating that mitophagy plays a key role in the regulation of CoQ0-inhibited NLRP3 inflammasome activation. Nrf2 knockdown significantly decreased IL1ß expression in LPS/ATP-stimulated RAW264.7 macrophages suggesting that CoQ0 inhibited ROS-mediated NLRP3 inflammasome activation and IL1ß expression was suppressed due to the Nrf2 activation. Hence, this study showed that CoQ0 might be a promising candidate for the therapeutics of inflammatory disorders due to its effective anti-inflammatory as well as antioxidant properties.

GRK3 as a Prognosis Biomarker in Gastric Cancer.

Background: Globally, gastric cancer is ranked 4th and 3rd in terms of incidence and mortality rate among all cancer types. This study aimed to examine the relationship between G protein-coupled receptor kinase 3 (GRK3) and gastric cancer prognosis and investigate the role of GRK3 in gastric cancer carcinogenesis. Methods: GRK3 level in gastric tissues and cells were determined using immunohistochemistry and immunoblotting. Kaplan-Meier analysis with the log-rank test was employed to evaluate the relationship between GRK3 expression and gastric cancer prognosis. RNAi technology was applied to examine the effects of GRK3 inhibition on gastric cancer proliferation and spread. Results: GRK3 overexpression was correlated significantly with lymphatic metastasis (P = 0.0011), distant metastasis (P < 0.0001), TNM stage (P = 0.0035), and vascular invasion (P = 0.0025). Kaplan-Meier survival analysis showed that the disease-free survival and overall survival of patients with high GRK3 expression were significantly shorter than those of patients with low GRK3 expression. Multivariate Cox regression analysis also showed that the overexpression of GRK3 was an independent prognostic biomarker of gastric cancer (P = 0.029). In cultured gastric cancer cells, GRK3 knockdown inhibited cell proliferation, migration, and invasion. Further analysis revealed that more GRK3-knockdown cells were in G0/G1 phase and few cells were in S phase, thereby inhibiting cell proliferation. Conclusions: GRK3 overexpression can be a candidate biomarker for gastric cancer prognosis. GRK3 is also a potential therapeutic target for gastric cancer.

Feasibility of single-port laparoscopic cholecystectomy using a homemade laparoscopic port: a clinical report of 50 cases.

AIM: To report the clinical experience of transumbilical single-port laparoscopic cholecystectomy (TUSPLC), using a homemade laparoscopic access port composed of two inexpensive and common pieces of equipment readily available in the operating room. METHODS: Fifty consecutive patients with gallstones, including ten patients (20%) with acute cholecystitis, underwent single-port laparoscopic cholecystectomy (LC) using a homemade single port composed of a segment of corrugated breathing tube and a pair of surgical gloves. The port was inserted into the umbilicus for simultaneous placement of multiple conventional instruments into the abdominal cavity. All patients underwent dome-down LC using traditional instruments with manually angulated shafts; dissection was done using electrocautery or harmonic scalpel. RESULTS: All but two procedures were completed uneventfully. Two patients with acute cholecystitis due to dense adhesions in the triangle of Calot necessitated conversion to two- and four-port laparoscopic procedures, respectively. Operative time averaged 73 ± 2 min for chronic cholecystitis and 95 ± 5 min for acute cholecystitis. There were no perioperative port-related or surgical complications, except for two patients who developed wound seroma and recovered after conservative treatment. We found that healing of the umbilical wound left virtually no scar in all patients. CONCLUSION: The homemade umbilical port reported in this study is useful for multiple instrument access and allows TUSPLC to be performed safely, with its inherent cosmetic and cost advantages. Further studies of this technique are ongoing.

Correction: Hseu, Y.-C., et al. The In Vitro and In Vivo Anticancer Properties of Chalcone Flavokawain B through Induction of ROS-Mediated Apoptotic and Autophagic Cell Death in Human Melanoma Cells. Cancers 2020, 12, 2936.

In the original article [...].