ZNF746 plays cardinal roles on colorectal cancer (CRC) cell invasion and migration and regulates mitochondrial dynamics and morphological changes of CRC cells-Role of combined melatonin and 5-FU regimen.

The underlying mechanism of colorectal cells developing into cancer cells has been extensively investigated, yet is still not fully delineated, resulting in the treatment of advanced colorectal cancer (CRC) remains regrettably an unmet need. Zinc Finger Protein 746/Parkin-interacting substrate (ZNF746/PARIS) has previously been identified to play a fundamental role on bladder cancer cell proliferation and metastasis that were effectively inhibited by melatonin (Mel). In this study, we utilized ex vivo/in vivo studies to verify whether the ZNF746 signaling was also crucial in CRC growth/invasion/migration. Tissue-bank specimens showed that the protein expression of ZNF746 was significantly increased in CRC than that of healthy colorectal tissues (p < 0.001). Additionally, in vitro study demonstrated that excessive expression of ZNF746 significantly inhibited mitochondrial activity via (1) interfering with the dynamic balance of mitochondrial fusion/fission and (2) inhibiting the protein expression of MFN1/MFN2/PGC1a (all p < 0.001). Furthermore, we identified that inhibition of ZNF746 protein expression significantly reduced the resistance of CRC cell lines to the anticancer drug of 5-FU (p < 0.001), whereas overexpression of ZNF746 significantly augmented resistance of CRC cells to 5-FU (all p < 0.001). Finally, using the cell culture method, we found that combined Mel and 5-FU was superior to merely one on promoting the CRC cell apoptosis (p < 0.001). Our results confirmed that ZNF746 signaling played a cardinal role of CRC cell proliferation/survival and combined Mel and 5-FU treatment attenuated the resistance of CRC cells to the drug mainly through suppressing this signaling.

Melatonin-Assisted Cisplatin Suppresses Urinary Bladder Cancer Cell Proliferation and Growth through Inhibiting PrPC-Regulated Cell Stress and Cell Proliferation Signaling.

This study investigated whether melatonin (Mel) would promote cisplatin to suppress the proliferation and growth of bladder cancer (BC) cells by inhibiting cellular prion protein (PrPC)-mediated cell stress and cell proliferation signaling. An immunohistochemical staining of tissue arrays from BC patients demonstrated that the PrPC expression was significantly upregulated from stage I to III BC (p < 0.0001). The BC cellline of T24 was categorized into G1 (T24), G2 (T24 + Mel/100 µM), G3 (T24+cisplatin/6 µM), G4 (PrPC overexpression in T24 (i.e., PrPC-OE-T24)), G5 (PrPC-OE-T24+Mel), and G6 (PrPC-OE-T24+cisplatin). When compared with a human uroepithelial cell line (SV-HUC-1), the cellular viability/wound healing ability/migration rate were significantly increased in T24 cells (G1) and further significantly increased in PrPC-OE-T24 cells (G4); and they were suppressed in Mel (G2/G5) or cisplatin (G3/G6) treatment (all p < 0.0001). Additionally, the protein expressions of cell proliferation (PI3K/p-Akt/p-m-TOR/MMP-9/PrPC), cell cycle/mitochondrial functional integrity (cyclin-D1/clyclin-E1/ckd2/ckd4/mitochondrial-cytochrome-C/PINK1), and cell stress (RAS/c-RAF/p-MEK1/2, p-ERK1/2) markers showed a similar pattern of cell viability among the groups (all p < 0.001). After the BC cell line of UMUC3 was implanted into nude mouse backs, by day 28 mthe BC weight/volume and the cellular levels of PrPC/MMP-2/MMP-9 were significantly, gradually reduced from groups one to four (all p < 0.0001). The protein expressions of cell proliferation (PI3K/p-Akt/p-m-TOR/MMP-9/PrPC), cell cycle/mitophagy (cyclin-D1/clyclin-E1/ckd2/ckd4/PINK1), and cell stress (RAS/c-RAF/p-MEK1,2/p-ERK1,2) signaling were significantly, progressively reduced from groups one to four, whereas the protein expressions of apoptotic (Mit-Bax/cleaved-caspase-3/cleaved-PARP) and oxidative stress/mitochondrial damaged (NOX-1/NOX-2/cytosolic-cytochrome-C/p-DRP1) markers expressed an opposite pattern of cell proliferation signaling among the groups (all p < 0.0001). Mel-cisplatin suppressed BC cell growth/proliferation via inhibiting the PrPC in upregulating the cell proliferation/cell stress/cell cycle signaling.

Cupping Therapy as an Adjunctive Therapy for Side Effects of Colorectal Cancer Treatment: A Prospective Observational Study.

Objective: The purpose of this study was to assess the effects of moving cupping therapy in people with colorectal cancer (CRC) experiencing chemotherapy-related side effects. Methods: A prospective observational study was conducted in people diagnosed with CRC who were treated for the side effects of their chemotherapy. Participants received cupping therapy 3 times a week for 10 consecutive weeks at our traditional Chinese medicine ward. Their quality of life and meridian energies were evaluated both at baseline and at 3 months after the treatment course. Results: Forty-six individuals with CRC were enrolled and 34 completed the study. The average number of cycles of chemotherapy during the study was 4.5. The mean number of moving cupping treatments was 25.7. After the moving cupping treatment program, participants exhibited significant improvements in quality of life, physical function, fatigue, nausea/vomiting, sleep disturbance, and pain. Conclusion: For the participants in this study, moving cupping therapy relieved some chemotherapy-related side effects and improved quality of life in people with CRC.

Melatonin and hyperbaric oxygen therapies suppress colorectal carcinogenesis through pleiotropic effects and multifaceted mechanisms.

Colorectal cancer (CRC) is the third most common cancer worldwide. Colorectal carcinogenesis is frequently induced by hypoxia to trigger the reprogramming of cellular metabolism and gain of malignant phenotypes. Previously, hyperbaric oxygen (HBO) therapy and melatonin have been reported to alter the hypoxic microenvironment, resulting in inhibiting cancer cell survival. Accordingly, this study tested the hypothesis whether HBO and melatonin effectively inhibited CRC carcinogenesis. In vitro results indicated that melatonin therapy significantly suppressed the malignant phenotypes, including colony formation, growth, invasion, migration and cancer stemness with dose-dependent manners in CRC cell lines through multifaceted mechanisms. Similar to in vitro study, in vivo findings further demonstrated the melatonin, HBO and combined treatments effectively promoted apoptosis (cleaved-caspase 3/ cleaved-PARP) and arrested tumor proliferation, followed by inhibiting colorectal tumorigenesis in CRC xenograft tumor model. Moreover, melatonin, HBO and combined treatments modulated multifaceted mechanisms, including decreasing HIF-1α expression, alleviating AKT activation, repressing glycolytic metabolism (HK-2/PFK1/PKM2/LDH), restraining cancer stemness pathway (TGF-ß/p-Smad3/Oct4/Nanog), reducing inflammation (p-NFκB/ COX-2), diminishing immune escape (PD-L1), and reversing expression of epithelial mesenchymal transition (E-cadherin/N-cadherin/MMP9). In conclusion, melatonin and HBO therapies suppressed colorectal carcinogenesis through the pleiotropic effects and multifaceted mechanisms, suggesting melatonin and HBO treatments could be novel therapeutic strategies for CRC treatment.

Combined high energy of extracorporeal shock wave and 5-FU effectively suppressed the proliferation and growth of tongue squamous cell carcinoma.

BACKGROUND: We tested the hypothesis that extracorporeal shock wave (ECSW)-assisted 5-FU therapy effectively suppressed human tongue squamous carcinoma cell line SAS (i.e., SAS cells) proliferation and tumor growth. METHODS AND RESULTS: In vitro study showed that as compared with lower ECSW energy (≤0.12 mJ/mm2), higher ECSW energy (≥0.25-035 mJ/mm2) significantly suppressed the SAS cell proliferation and upregulated tumor cell apoptosis/DNA-damage/oxidative-stress, whereas combined higher ECSW energy (0.35 mJ/mm2) and 5-FU (20uM) further significantly altered the expressions of these parameters (all p < 0.001). Adult male nude mice (NM) (n = 36) were equally categorized into group 1 (2.0 × 105 SAS cells were implanted into NM back), group 2 [SAS in NM back + stepwise-increased ECSW energy (from 0.05/0.1/0.3/to 0.5 mJ/mm2)/500 impulses which applied to the tumor at days 9/12/15/21], group 3 (SAS in NM back + 5-FU/i.p./7 mg/kg/every 3-day) and group 4 (SAS in NM back + ECSW + 5-FU) and tumors were removed from each animal by day-28. The result showed that tumor volume and tumor weight were significantly progressively reduced from group 1 to group 4 (all p < 0.0001). The protein expressions of apoptotic (mitochondrial-Bax/cleaved-caspase3/cleaved-PARP/cyclophyllin-D), autophagic (ratio of LC3B-II/LC3B-I) and oxidative-stress (NOX-1/NOX-2) biomarkers displayed an opposite pattern of tumor mass among the groups, whereas the cell-stress signaling (p-PI3K/p-Akt/p-m-TOR, and ASK1/MKK4/MKK7/p38/p-JNK/p-c-JUN), MAP kinase family members (RAS/cRAF/KRAS/BRAF/p-ERK1/2), tumor protein (p53) and cellular levels of angiogenesis/DNA-damage (α-SMA+/VEGF+/γ-H2AX+) exhibited an identical pattern of tumor mass among the groups (all p < 0.0001). CONCLUSION: Combined high-energy ECSW and 5-FU offers an additional benefit for suppressing the cancer cell proliferation and tumor growth.

CHD4 as an important mediator in regulating the malignant behaviors of colorectal cancer.

Colorectal cancer (CRC) has ranked first in terms of incidence in Taiwan. Surgical resection combined with chemo-, radio-, or targeted-therapies are the main treatments for CRC patients in current clinical practice. However, many CRC patients still respond poorly to these treatments, leading to tumor recurrence and an unacceptably high incidence of metastasis and death. Therefore, appropriate diagnosis, treatment, and drug selection are pressing issues in clinical practice. The Mi-2/nucleosome remodeling and deacetylase complex is an important epigenetic regulator of chromatin structure and gene expression. An important component of this complex is chromodomain-helicase-DNA-binding protein 4 (CHD4), which is involved in DNA repair after injury. Recent studies have indicated that CHD4 has oncogenic functions that inhibit multiple tumor suppressor genes through epigenetic regulation. However, the role of CHD4 in CRC has not yet been well investigated. In this study, we compared CHD4 expression in CRC patients from The Cancer Genome Atlas database. We found higher levels of CHD4 expression in CRC patients. In a series of in vitro experiments, we found that CHD4 affected cell motility and drug sensitivity in CRC cells. In animal models, the depletion of CHD4 affected CRC tumor growth, and the combination of a histone deacetylase 1 (HDAC1) inhibitor and platinum drugs inhibited CHD4 expression and increased the cytotoxicity of platinum drugs. Moreover, CHD4 expression was also a prognostic biomarker in CRC patients. Based on the above results, we believe that CHD4 expression is a viable biomarker for predicting metastasis CRC patients, and it has the potential to become a target for drug development.

Jagged2 progressively increased expression from Stage I to III of Bladder Cancer and Melatonin-mediated downregulation of Notch/Jagged2 suppresses the Bladder Tumorigenesis via inhibiting PI3K/AKT/mTOR/MMPs signaling.

Background: This study assessed the expression of Jagged2 in human bladder cancer (BC) tested the hypothesis that melatonin (Mel) inhibited the tumorigenesis of BC cells mainly through downregulating the Notch/Jagged2 and PI3K/AKT/mTOR/MMPs(2&9) signaling pathways. Methods and Results: Tissue array from BC patients showed that the gene and protein expressions of JAG2/Jagged2 were significantly upregulated from T1 to T3 (primary tumor size) and from stage I to III (all p<0.001). In vitro study showed that in BC cell line of UMUC3, the cellular and protein expressions of Jagged2 were significantly attenuated in Mel-treated UMUC3 and further attenuated in UMUC3 shRNA silenced Notch/JAG2 (UMUC3KD) than in UMUC3 only (all p<0.0001). The protein expressions of Notch/Jagged2/MMPs(2&9)/PI3K/p-AKT/mTOR/p53/ratio of LC3BII/LC3B-I were significantly progressively reduced from UMUC3 to UMUC3+Mel/1.0mM, further to UMUC3+Mel/2.0mM and furthermore to UMUC3KD (all p<0.0001). The cell proliferation/invasion/colony formation/healing-process were significantly inhibited in Mel-treated/2.0mM UMUC3 and further significantly inhibited in UMUC3KD regardless of Mel treatment as compared with UMUC3 only (all p<0.0001). By day 28 after UMUC3 implanted into nude mouse back, the BC weight/volume were significantly reduced in UMUC3+Mel (100 mg/kg/day) and furthermore reduced in UMUC3KD (all p<0.0001) as compared with UMUC3 only (all p<0.0001). The cellular (MMPs(2&9)/Notch/Jagged2) and protein (Notch/Jagged2/PI3K/p-AKT/mTOR/MMPs(2&9)) exhibited a similar trend, whereas the PTEN protein level exhibited an opposite pattern of PI3K among three groups (all p<0.0001). Conclusion: Notch/Jagged-PI3K/p-AKT/mTOR/MMPs is one essential signaling pathway for BC survival, proliferation and invasion that were remarkably suppressed by Mel treatment.

Level and Value of T Cell-derived Circulating Microparticles in Liver Cirrhosis Patients.

BACKGROUND/AIM: We examined the hypothesis that T cell-derived-circulating microparticles (MPs) are increased in liver-cirrhosis (LC) patients compared to normal subjects and are also increased in chronic hepatitis compared to acute-decompensated-liver cirrhosis (ADLC). PATIENTS AND METHODS: A total of 66 LC patients, including 35 with ADLC and 31 with non-decompensated-LC (NDLC), were enrolled in the study. Ten volunteers served as controls. RESULTS: Flow-cytometric analysis showed that circulating levels of T-cell derived MPs (i.e., total MPs and CD4+/CD8+/CD54+MPs) were higher in LC patients than in the controls (all p<0.003). Total MPs and CD8+MPs were higher in NDLC than in ADLC patients. There were good correlations between CD8+MPs and ADLC as well as between total MPs and chronic hepatitis. Multivariate-linear-regression analysis showed that NDLC was independently predictive of increased circulating CD8+MPs levels (p<0.05) and chronic hepatitis independently predictive of increased circulating total MPs levels (p<0.001)/CD4+MPs (p<0.05). CONCLUSION: Circulating levels of T-cell-derived MPs were increased in ADLC patients and were even more elevated in NDLC patients compared to healthy-control subjects.

Adipose-derived mesenchymal stem cell-derived exosomes markedly protected the brain against sepsis syndrome induced injury in rat.

This study tested the hypothesis that sepsis syndrome [SS-induced by cecal-ligation and puncture (CLP)]-induced systemic inflammation and brain damage in rats were effectively suppressed by allogenic adipose-derived mesenchymal stem cell-derived exosome (AMSCEXO). SD rats (n = 72) were divided into group 1 [sham-control (SC)], group 2 (SS only) and group 3 (SS + AMSCEXO) and equally euthanized at 6/24/48/72 h after SS induction, respectively. By 6/16/24/72 h, flow cytometric analyses demonstrated the numbers of inflammatory cells (Ly6G+/CD11b/c+), immune (CD3+/CD4+ cells/CD3+/CD8+ cells) and early (AN-V+/PI-)/late (AN-V+/PI+) apoptotic cells in circulation were significantly increased in group 2 than in groups 1 and 3, and significantly increased in group 3 than in group 1, whereas the number of T-reg+ cells was significantly progressively increased from groups 1 to 3 (all P < 0.0001). At 6/16/24/72 h, the numbers of (CD3+/CD4+ cells/CD3+/CD8+ cells/T-reg+ cells) in spleen exhibited an identical pattern of circulation among the three groups (all P < 0.0001). ELISA showed inflammatory mediators (IL-6/TNF-α) in circulating/cerebrospinal fluid at 6/24/72 h displayed an identical trend as the immune cells among the three groups (all P < 0.0001). Microscopic findings demonstrated that the cellular expressions of inflammatory (F4/80+//MMP-9+//CD14+//GFPA+) and brain-damaged (AQP4+/γ-H2AX+) biomarkers at 24/72 h exhibited an identical pattern of immune cells among the three groups (all P < 0.0001). The protein expressions of inflammatory (IL-1ß/MMP-9/TNF-α/NF-κB/TLR2/TLR-4/MyD88/HMGB1), apoptotic (cleaved-caspase3/PARP/mitochondrial-Bax) and oxidative-stress (NOX-1/NOX-2/oxidized protein) biomarkers displayed an identical pattern as the immune cells among the three groups (all P < 0.0001). In conclusion, SS elicited vigorously inflammatory reaction not only in circulation but also in spleen/brain, resulting in serious brain damage.

Synergistic effect of combined melatonin and adipose-derived mesenchymal stem cell (ADMSC)-derived exosomes on amelioration of dextran sulfate sodium (DSS)-induced acute colitis.

This study tested the hypothesis that melatonin (Mel) and adipose-derived mesenchymal stem cell-derived exosomes effectively suppress dextran sulfate sodium (DSS)-induced acute inflammatory colitis (AIC) in rats. To determine whether Mel-exosome treatment could ameliorate the severity of AIC, we treated Sprague Dawley rats with DSS-induced AIC with Mel, exosomes, or combined Mel-exosome therapy and evaluated the effects on AIC. First, to induce an inflammatory response in vitro, we treated HT-29 cells with lipopolysaccharide (LPS) and evaluated the response to Mel and/or exosome treatment. We found that expression of NOX-1, NOX-2, MMP-9, NF-κB, iNOS, ICAM-1, and COX-2 was significantly higher in HT-29 cells treated with LPS than in control cells, and was significantly reduced by either exosome or Mel treatment (P<0.001 for all). In vivo, flow cytometric analysis showed that, compared to untreated rats with AIC, the number of circulating inflammatory cells was lowest in rats treated with combined Mel-exosome treatment than in rats treated with either Mel or exosomes alone (P<0.0001). Compared with controls, as well as Mel or exosome treatment alone, combined Mel-exosome treatment ameliorated the effects of DSS-induced AIC as evidenced by changes in the expression of markers for inflammation, oxidative stress, apoptosis, and fibrosis (P<0.0001 for all). Additionally, histopathological findings showed that colon injury score, expression of inflammatory and DNA-damage markers, and bloody stool were all improved following combined Mel-exosome treatment (P<0.0001 for all). In conclusion, combined Mel-exosome treatment significantly protected the rat colon against DSS-induced AIC injury.

Long-Term Consequences of Nonclosure of Mesenteric Defects after Traditional Right Colectomy.

BACKGROUND: There are still discrepancies among general/colorectal surgeons regarding closure of mesenteric defect in scientific literature. This study aimed to assess the long-term consequences of nonclosure of the mesenteric defect after open right colectomy. METHODS: A 7-year retrospectively collected and continuous database revealed 212 consecutive patients who had undergone traditional right colectomy without closing the mesenteric defects at Kaohsiung Chung-Gung Memorial Hospital; all patients were operated by a single surgeon. Among these patients, 17 were excluded (those who died within 30 days after surgery or those who received an end ileostomy). The mean age of the 195 patients (58% men and 42% women) was 61.6 ± 12.6 years, and the follow-up period was 4.1 ± 2.8 years (interquartile range 0.09 ~ 10.4). RESULTS: Forty-four patients (22.5%) encountered intestinal obstruction. Nine (20.4%) required surgical intervention. The cause of intestinal obstruction was adhesion (n=1), ventral hernia (n=1), and cancer recurrence (n=7). Conservative treatment was successful in 35 patients. The intestinal obstruction group (n = 44) were similar to the no-intestinal obstruction group (n = 151) in terms of the following parameters: age, sex, previous abdominal surgery, indication for colectomy, and procedure related complications. Carcinomatosis was found to increase the incidence of intestinal obstruction. No patient developed intestinal obstruction because of the nonclosure of mesenteric defects after right colectomy. CONCLUSION: This study suggested that routine procedure of closing the mesenteric defect after open right colectomy might not be beneficial. Additional studies with extended long-term follow-up periods are needed to confirm the benefits of the nonclosure.

Adipose-derived mesenchymal stem cell-derived exosomes alleviate overwhelming systemic inflammatory reaction and organ damage and improve outcome in rat sepsis syndrome.

This study tested the hypothesis that healthy adipose-derived mesenchymal stem cell (ADMSC)-derived exosomes (HMSCEXO) and apoptotic (A) (induced by 12 h hypoxia/12 h starvation)-ADMSC-derived exosomes (AMSCEXO) were comparably effective at alleviating sepsis syndrome [SS; induced by cecal-ligation and puncture (CLP)]-induced systemic inflammation and reduced organ damage and unfavorable outcomes in rats. SD rats were divided into sham control (SC), SS only, SS + HMSCEXO (100 µg intravenous administration 3 h after CLP), and AMSCEXO. By day 5 after CLP procedure, the mortality rate was significantly higher in SS than in SC and HMSCEXO (all P < 0.01), but it showed no significant different between SC and HMSCEXO, between AMSCEXO and HMSCEXO or between SS and AMSCEXO (P > 0.05). The levels of inflammatory mediators in circulation (CD11b/c/Ly6G/MIF), bronchioalveolar lavage (CD11b/c/Ly6G) and abdominal ascites (CD11b/c/CD14/Ly6G/MIF) were highest in SS, lowest in SC and significantly higher in AMSCEXO than in HMSCEXO (all P < 0.001). The circulating/splenic levels of immune cells (CD34+/CD4+/CD3+/CD8+) were expressed in an identical pattern whereas the T-reg+ cells exhibited an opposite pattern of inflammation among the groups (all P < 0.001). The protein expressions of inflammation (MMP-9/MIF/TNF-α/NF-κB/IL-1ß) and oxidative stress (NOX-1/NOX-2/oxidized protein), and cellular expressions (CD14+/CD68+) in lung/kidney parenchyma exhibited an identical pattern of inflammatory mediators (all P < 0.001). The kidney/lung injury scores displayed an identical pattern of inflammatory mediators among the groups (all P < 0.001). In conclusion, HMSCEXO might be superior to AMSCEXO for improving survival and suppressing the inflammatory reactions in rats after SS.

Extracorporeal shock wave markedly alleviates radiation-induced chronic cystitis in rat.

This study tested the hypothesis that extracorporeal shock wave (ECSW) treatment can effectively inhibit radiation-induced chronic cystitis (CC). Adult male Sprague-Dawley (SD) rats (n = 24) were randomly divided into group 1 (normal control), group 2 (CC induced by radiation with 300 cGy twice with a four-hour interval to the urinary bladder), group 3 [CC with ECSW treatment (0.2 mJ/mm2/120 impulses/at days 1, 7, and 14 after radiation)]. Bladder specimens were harvested by day 28 after radiation. By day 28 after radiation, the degree of detrusor contraction impairment was significantly higher in group 2 than that in groups 1 and 3, and significantly higher in group 3 than that in group 1 (P<0.0001). The urine albumin concentration expressed an opposite pattern compared to that of detrusor function among the three groups (P<0.0001). The bladder protein expressions of inflammatory (TLR-2/TLR-4/IL-6/IL-12/MMP-9/TNF-α/NF-κB/RANTES/iNOS) and oxidative-stress (NOX-1/NOX-2/oxidized protein) biomarkers exhibited a pattern identical to that of urine albumin in all groups (all P<0.0001). The cellular expressions of inflammatory (CD14+/CD68+/CD74+/COX-2/MIF+/substance P+) and cytokeratin (CK13+/HMW CK+/CK+17/CK+18/CK+19) biomarkers, and collagen-deposition/fibrotic areas as well as epithelial-damaged score displayed an identical pattern compared to that of urine albumin among the three groups (all P<0.0001). In conclusion, ECSW treatment effectively protected urinary bladder from radiation-induced CC.

CHD4-mediated loss of E-cadherin determines metastatic ability in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a subtype of cancer with aggressive behaviors (high recurrence and metastasis rate) and poor prognosis. Therefore, studying the determining factors that lead to malignant TNBCs is necessary to develop personalized therapy and improve survival rates. In this study, we first analyzed levels of chromodomain helicase DNA binding protein 4 (CHD4) in 60 TNBC patients by immunohistochemical staining. We then clarified the role of CHD4 in TNBC and non-TNBC cell lines. Our clinical data indicated that higher CHD4 expression is positively correlated with metastatic stage, tumor recurrence, and survival status. Consistent with the clinical analytical data, our in vitro data also indicated that high level of CHD4 is positively correlated with malignant behaviors in TNBC cells, such as cell motility and mortality. For further analyses, we found that E-cadherin, N-cadherin and fibronetin are involved in CHD4-mediated epithelial-mesenchymal transition (EMT). Silencing of CHD4 also increased drug sensitivity to cisplatin and PARP1 inhibitor, especially in TNBC cells. Altogether, our findings showed that CHD4 is not only a potential prognostic biomarker for TNBC patient survival, but is also a powerful candidate in the development of new anti-cancer agents in TNBC.

Therapeutic effect of rosuvastatin and propylthiouracil on ameliorating high-cholesterol diet-induced fatty liver disease, fibrosis and inflammation in rabbit.

This study tested the hypothesis that high-cholesterol diet (HCD)-induced fatty liver disease could be ameliorated by rosuvastatin (Ros) and propylthiouracil (PTU) therapy. Thirty-two Zealand rabbits were equally divided into group 1 (sham-control), group 2 (HCD for 8 weeks), group 3 [HCD-Ros (20 mg/kg/day administration after 4-week HFD for 4 weeks)], group 4 [HCD-PTU (0.1% PTU in drinking water) with treatment course as group 3]. Liver weight, fibrosis, collagen deposition area, and serum levels of AST/ALT were highest in group 2, lowest in group 1, and significantly higher in group 4 than group 3 (all P<0.0001). The levels of inflammatory (TNF-α/NF-κB/IL-1ß/IL-6/MMP-9/VCAM-1/PAI-1/TLR-4, MyD88/IL-12/IFN-γ), oxidative stress (NOX-1/NOX-2/oxidized protein), apoptotic (Bax/cleaved-capase-3/PARP), fibrotic (Smad-3/TGF-ß), and mitochondria-damaged (cytosolic-cytochrome-C) proteins showed an identical pattern, whereas antiapoptotic (Bcl-2), mitochondrial-integrity (mitochondrial-cytochrome-C) and antioxidative (SIRT1/SIRT3) biomarkers exhibited an opposite pattern to fibrosis among the four groups (all P<0.0001). The cellular expressions of inflammatory (Kupffer/CD14/CD44), α-fetoprotein-positively stained biomarkers, apoptotic nuclei and fat cells displayed an identical pattern to fibrosis (all P<0.0001). In conclusion, Ros-PTU therapy attenuated liver fibrosis, inflammatory reaction and generation of oxidative stress and fatty liver after HCD challenge in rabbits.

Melatonin treatment enhances therapeutic effects of exosomes against acute liver ischemia-reperfusion injury.

This study tests the hypothesis that combined melatonin and exogenic adipose mesenchymal stem cell (ADMSC)-derived exosome treatment offers superior protection against liver ischemia-reperfusion (LIR) injury compared to either alone. In vitro studies utilized a macrophage cell line (RAW) pretreated with lipopolysaccharide and hepatocytes pretreated with melatonin or exosomes before hypoxia treatment, while in vitro experiments involved analyses of liver specimens from male adult Sprague-Dawley rats (n = 50) equally categorized into sham controls (SC), LIR only, LIR-exosome (100 µg, 30 minute post-LIR), LIR-melatonin (20 mg/kg, 30 minute post-LIR and 50 mg/kg at 6 and 18 hours post-LIR), and LIR-exosome-melatonin groups. In vitro studies showed suppression of inflammation (MIF, MMP-9, IL-1ß, TNF-α, COX-2) and oxidative stress (NOX-1, NOX-2, oxidized protein)/apoptosis (cleaved caspase 3 and PARP) by exosome and exosome/melatonin treatment, respectively (all P<0.001). In vivo data demonstrated lowest liver injury score and plasma AST concentrations in LIR-exosome-melatonin group compared with other groups (P<0.001). Besides, expressions of inflammatory markers at protein (ICAM-1, IL-1ß, MMP-9, TNF-α, NF-κB, RANTES) and cellular (CD3+, CD4+, CD8+, CD161+, CD11+, CD14+, F4/80) levels, and protein expressions of apoptosis (cleaved caspase-3, PARP), oxidative stress (NOX-1, NOX-2), DNA damage (γ-H2AX) and mitochondrial damage (cytosolic cytochrome-C) markers displayed a pattern similar to that of liver injury score, whereas protein expression of anti-oxidants (HO-1, NQO-1) showed progressive increase from SC to the combined treatment group (all P<0.001). In conclusion, combined exosome-melatonin regimen was superior to either alone in protecting the liver against ischemia-reperfusion injury.

Human lung cancer-derived microparticles enhanced angiogenesis and growth of hepatoma cells in rodent lung parenchyma.

This study tested the hypothesis that human lung cancer-derived microparticles (LcD-MPs) played an important role in tumor angiogenesis and growth. Fischer 344 rats (F344, n=18) were equally categorized into group 1 [Sham Control (3.0 mL normal saline intravenous injection (IV))], group 2 [hepatoma cell line (2.0 x 10(6) cells, IV)], and group 3 [hepatoma cell line + LcD-MPs (3.0 x 10(6), IV)]. Animals were euthanized by day 28 after hepatoma cells transfusion. Our result showed that the gross pathology confirmed growth of hepatoma cell line in lung parenchyma. The size and weight of the lungs were significantly increased in group 2 and further elevated in group 3 than in group 1 (all p<0.001). Histopathological analysis demonstrated that the lung crowded score and number of small vessel exhibited an identical pattern, whereas the number of alveolar sacs showed an opposite pattern compared to that of total lung weight among the three groups (all p<0.0001). The cellular expressions of CD34(+), CXCR4(+), c-Kit(+), CK19(+), VEGF(+) and vimentin+ cells in lung parenchyma exhibited an identical pattern compared to those of total lung weight among all groups (all p<0.001). The protein expressions of apoptotic (Bax, cleaved caspase-3 and c-PARP), fibrotic (Smad3, TGF-ß), and tumor suppression (PTEN) biomarkers showed an identical pattern, whereas that of anti-apoptotic (Bcl-2) and anti-fibrotic (Smad1/5, BMP-2) biomarkers were displayed an opposite pattern compared to that of total lung weight among all groups (all p<0.001). The MPs could enhance angiogenesis and accelerated hepatoma cell growth in rodent lung parenchyma.

Combination of adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes for protecting kidney from acute ischemia-reperfusion injury.

BACKGROUND: In this study, we tested the hypothesis that a combined adipose-derived mesenchymal stem cell (ADMSC) and ADMSC-derived exosome therapy protected rat kidney from acute ischemia-reperfusion (IR) injury (i.e., ligation of both renal arteries for 1h and reperfusion for 72h prior to euthanization). METHODS AND RESULTS: Adult-male SD rats (n=40) were equally categorized into group 1 (sham control), group 2 (IR), group 3 [IR+exosome (100µg)], group 4 [IR+ADMSC (1.2×10(6) cells)], and group 5 (IR-exosome-ADMSC). All therapies were performed at 3h after IR procedure from venous administration. By 72h, the creatinine level and kidney injury score were the lowest in group 1 and the highest in group 2, significantly higher in group 3 than in groups 4 and 5, and significantly higher in group 4 than in group 5 (all P<0.0001). The protein expression of inflammatory (TNF-α/NF-κB/IL-1ß/MIF/PAI-1/Cox-2), oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (Bax/caspase-3/PARP), and fibrotic (Smad3/TGF-ß) biomarkers showed an identical pattern, whereas the anti-apoptotic (Smad1/5, BMP-2) and angiogenesis (CD31/vWF/angiopoietin) biomarkers and mitochondrial cytochrome-C showed an opposite pattern of creatinine level among the five groups (all P<0.001). The microscopic findings of glomerular-damage (WT-1), renal tubular-damage (KIM-1), DNA-damage (γ-H2AX), inflammation (MPO/MIF/CD68) exhibited an identical pattern, whereas the podocyte components (podocin/p-cadherin/synaptopodin) displayed a reversed pattern of creatinine level (all P<0.0001). CONCLUSION: Combined exosome-ADMSC therapy was superior to either one for protecting kidney from acute IR injury.

Combined melatonin and exendin-4 therapy preserves renal ultrastructural integrity after ischemia-reperfusion injury in the male rat.

We tested whether combined melatonin (Mel) and exendin-4 (Ex4) treatment can better preserve glomerular structural integrity after ischemia-reperfusion (IR) injury compared with either alone. Adult male Sprague Dawley rats (n = 50) were equally divided into sham control (SC), IR, IR-Ex4 (10 µg/kg subcutaneously 30 min after reperfusion and daily for 5 days), IR-Mel (20 mg/kg intraperitoneally at 30 min postreperfusion and 50 mg/kg at 6 and 18 hr), and IR-Ex4-Mel were euthanized at day 14. Serum creatinine level and urine protein-to-creatinine ratio at days 3 and 14 were highest in IR group and lowest in SC, significantly higher in IR-Ex4 and IR-Mel groups than in IR-Ex4-Mel group (all P < 0.001) without significant difference between IR-Ex4 and IR-Mel groups. Changes in podocyte injury score (PIS) and kidney injury score were highest in IR group and lowest in SC, significantly higher in IR-Ex4 and IR-Mel groups than in IR-Ex4-Mel, and significantly higher in IR-Mel group than in IR-Ex4 group (all P < 0.001). Immunohistochemical microscopic findings of the expressions of FSP-1 and WT-1 (two glomerular damage indicators) and KIM-1 and snail (two renal tubular-damaged indicators) showed an identical pattern, whereas the expressions of ZO-1, p-cadherin, podocin, dystroglycan, fibronectin, and synaptopodin (six indices of glomerular integrity) demonstrated an opposite pattern compared to that of PIS among five groups (all P < 0.001). Protein expressions of inflammatory (TNF-α/NF-κB/MMP-9) and oxidative stress (NOX-1, NOX-2, oxidized protein) biomarkers exhibited an identical pattern to that of PIS among five groups (all P < 0.001). Combined melatonin-exednin-4 therapy further protected glomerulus from IR injury.

Protective effect of melatonin-supported adipose-derived mesenchymal stem cells against small bowel ischemia-reperfusion injury in rat.

We tested the hypothesis that combined melatonin and autologous adipose-derived mesenchymal stem cells (ADMSC) was superior to either alone against small bowel ischemia-reperfusion (SBIR) injury induced by superior mesenteric artery clamping for 30 min followed by reperfusion for 72 hr. Male adult Sprague Dawley rats (n = 50) were equally categorized into sham-operated controls SC, SBIR, SBIR-ADMSC (1.0 × 10(6) intravenous and 1.0 × 10(6) intrajejunal injection), SBIR-melatonin (intraperitoneal 20 mg/kg at 30 min after SI ischemia and 50 mg/kg at 6 and 18 hr after SI reperfusion), and SBIR-ADMSC-melatonin groups. The results demonstrated that the circulating levels of TNF-α, MPO, LyG6+ cells, CD68+ cells, WBC count, and gut permeability were highest in SBIR and lowest in SC, significantly higher in SBIR-ADMSC group and further increased in SBIR-melatonin group than in the combined therapy group (all P < 0.001). The ischemic mucosal damage score, the protein expressions of inflammation (TNF-α, NF-κB, MMP-9, MPO, and iNOS), oxidative stress (NOX-1, NOX-2, and oxidized protein), apoptosis (APAF-1, mitochondrial Bax, cleaved caspase-3 and PARP), mitochondrial damage (cytosolic cytochrome C) and DNA damage (γ-H2AX) markers, as well as cellular expressions of proliferation (PCNA), apoptosis (caspase-3, TUNEL assay), and DNA damage (γ-H2AX) showed an identical pattern, whereas mitochondrial cytochrome C exhibited an opposite pattern compared to that of inflammation among all groups (all P < 0.001). Besides, antioxidant expressions at protein (NQO-1, GR, and GPx) and cellular (HO-1) levels progressively increased from SC to the combined treatment group (all P < 0.001). In conclusion, combined melatonin-ADMSC treatment offered additive beneficial effect against SBIR injury.