Clinical characteristics and management of orbital apex syndrome: a 10-year multicentre experience.

BACKGROUND: Orbital apex syndrome (OAS) is a condition characterised by lesions within the orbital apex, leading to various ophthalmologic symptoms. This study aimed to analyse the clinical characteristics and treatment strategies of OAS with respect to aetiology. METHODS: This retrospective analysis utilised data from 5 medical institutions between 2013 and 2022. Patients who were diagnosed with OAS were initially enrolled, but patients who failed to follow up at least 1 month were excluded. The prevalence of initial ophthalmologic symptoms and visual improvement after treatment was compared according to aetiology. Factors related to visual improvement were analysed. RESULTS: Among 73 enrolled patients, the leading aetiology was tumours, followed by fungal infections and inflammation. Visual impairment and proptosis were prevalent in tumour-related OAS cases. Inflammation-related OAS exhibited a higher likelihood of painful eye movements and ophthalmoplegia. Ptosis was most frequently observed in fungal infection-related OAS. Notably, fungal infections emerged as the sole significant factor negatively impacting vision progression. In inflammation-related OAS, the time interval between symptom onset and the administration of steroids was longer in patients without visual improvement, even though there was no statistically significant difference. CONCLUSIONS: Tumours were the predominant cause of OAS. Visual impairment was a common manifestation in tumour-related OAS, while fungal infections were strongly associated with a poor visual prognosis. The timely administration of steroids might be helpful for improving vision in patients with inflammation-related OAS. However, further studies are needed to enhance understanding and management of OAS.

[Clinical value of focused ultrasound ablation surgery in the treatment of abdominal wall endometriosis].

Objective: To explore the effectiveness and safety of focused ultrasound ablation surgery (FUAS) for abdominal wall endometriosis. Methods: From November 2019 to October 2022, a total of 34 patients with abdominal wall endometriosis who underwent FUAS were collected, and their clinical features, imaging features, intraoperative treatment and side effects after treatment were analyzed retrospectively, and the improvement of symptoms and re-intervention were followed up. Results: (1) Characteristics of clinical data: the average age of 34 patients with abdominal wall endometriosis was (32.8±3.8) years old. The largest diameter of the lesion was 48 mm, and the median lesion diameter was 24 mm. Thirty cases (88%, 30/34) had moderate to severe periodic pain in abdominal incision before FUAS. All patients were diagnosed by preoperative magnetic resonance imaging, including 19 cases (56%, 19/34) of superficial type, 8 cases (24%, 8/34) of intermediate type and 7 cases (21%, 7/34) of deep type. (2) FUAS treatment parameters: ablation was completed with average operation time of (64±18) minutes, average sonication time was (385±108) s, (103±11) W of average power, (38 819±16 309) J of average total energy, the average treatment area volume of (3.11±1.42) cm3, and (377.79±106.34) s/h of average treatment intensity. (3) Efficiency: the pain of patients after FUAS was significantly relieved, and the pain scores of patients after 1 month, 3 months, 6 months and 1 year after FUAS were significantly decreased (Z=-4.66, -5.13, -5.11 and -4.91, all P<0.01). One year after FUAS, the near relief and effective pain relief rate was 74% (25/34), and the clinical effective rate was 85% (29/34). Five patients recurred after one year, including 3 patients who underwent abdominal wall endometriosis lesion resection and 2 patients who received drug treatment. One month after FUAS, the size of the lesion did not change significantly compared with that before FUAS (P>0.05), and the size of the lesion decreased significantly after FUAS at 3 months, 6 months and 1 year (Z=-2.15, -2.67 and -3.41, all P<0.05). It has no difference in pain relief among different types (P>0.05), but has significant difference in focus reduction among three types (P<0.01). (4) Safety: there were 34 cases (100%, 34/34) of skin burning sensation, 19 cases (56%, 19/34) of pain in the treatment area and 2 cases (6%, 2/34) of hematuria. All patients got better after corresponding treatments. Conclusion: FUAS is safe and effective for the treatment of abdominal wall endometriosis, which has clinical application value.

Comparison of sentinel lymph node biopsy and elective neck dissection for early oral cavity squamous cell carcinoma patients with clinically node-negative necks: systematic review and meta-analysis.

OBJECTIVE: This study aimed to compare the prognostic utility of sentinel node biopsy and elective neck dissection in early stage clinically node-negative oral cavity squamous cell carcinoma patients. METHOD: PubMed, Scopus, Embase, Web of Science and Cochrane Library databases were searched up to March 2022. Hazard ratios, Kaplan-Meier curves, p-values and survival outcomes were extracted. RESULTS: Twelve studies involving 10 583 patients were included. No significant differences in overall survival between sentinel node biopsy and elective neck dissection groups were found. Heterogeneity was not detected in pooled overall survival, disease-free survival and disease-specific survival analyses (all I2 less than 50). In subgroup analyses by follow-up period, sentinel node biopsy and elective neck dissection had similar prognostic value. CONCLUSION: Sentinel node biopsy might be a valuable alternative to elective neck dissection for the management of early stage clinically node-negative oral cavity squamous cell carcinoma.

[Pregnancy outcomes in patients with adenomyosis with fertility requirements: retrospective analysis of clinical data from real world].

Objective: To retrospectively analyze the pregnancy outcomes of patients with adenomyosis requiring fertility in a single center under real world condition. Methods: From June 2015 to May 2020, 231 cases of pregnancy complicated with adenomyosis diagnosed by ultrasound with fertility requirements were treated in the Women's and Children's Hospital Affiliated to Qingdao University with complete clinical data. And they were divided into three groups according to the treatment of adenomyosis before pregnancy: expectation group, drug group and operation group. The relevant data before pregnancy of the three groups were analyzed, and the pregnancy outcomes of the patients were summarized. According to whether the early pregnancy was treated with medication, the patients who were naturally conceived without symptoms of threatened abortion were divided into observation group and fetus protection group, and the pregnancy outcomes of the two groups were compared. Results: (1) Compared with the expectation group, the ages of patients in the drug group and the operation group were larger [(31.5±1.8) vs (34.1±3.7) vs (36.9±3.6) years old], and the difference was statistically significant (P<0.05). Only 9 patients (11.5%, 9/78) had clinical symptoms in the expectation group, while the patients in the drug group and the operation group had a higher proportion of dysmenorrhea and increased menstrual volume. The uterine volume of the drug group and the operation group were larger than that of the expectation group [(151±46) vs (166±27) vs (97±18) cm3], the difference was statistically significant (P<0.05). 78.6% (33/42) of the operation group were focal adenomyosis. The proportion of natural pregnancy in the expectation group was 97.4% (76/78), and in vitro fertilization and embryo transfer was mainly used in the drug group and the operation group. (2) The abortion rates of the three groups were 48.7% (26/111), 4/17, 67.5% (27/78) respectively. Compared with the drug group and the operation group, the preterm birth rate was lower [55.9% (33/111) vs 11/17 vs 12.5% (5/78)] and the natural delivery rate was higher [44.1% (26/111) vs 4/17 vs 67.5% (27/78)] in the expectation group. (3) There were 89 cases of spontaneous pregnancy without threatened abortion symptoms, including 31 cases in the observation group and 58 cases in the fetus protection group. Compared with the observation group, the abortion rate of patients in the fetus protection group was lower [41.9% (13/31) vs 34.5% (20/58)], and the difference was statistically significant (P<0.05). Conclusions: Patients with adenomyosis who have fertility requirements should be comprehensively evaluated and individualized treatment plans should be given. Pregnancy patients with adenomyosis have a high rate of miscarriage, and they should be included in the management of high-risk pregnant women. Active fetal protection treatment during early pregnancy might improve pregnancy outcomes.

Long non-coding RNA LINP1 functions as an oncogene in endometrial cancer progression by regulating the PI3K/AKT signaling pathway.

OBJECTIVE: Endometrial cancer (EC) accounts for about 6% of new cancer cases in female and about 3% of cancer-related deaths were caused by EC. The poor prognosis is mainly due to the distant spread and poor differentiation. In the current study, we want to figure out the role of long non-coding RNA (LncRNA) LINP1 in EC progression. PATIENTS AND METHODS: The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay was involved to access the expression level of LINP1 in EC cell lines and tissues. The Cell Counting Kit-8 (CCK-8) assay, colony formation assay, transwell and Matrigel assay were recruited to figure out the ability of LINP1 in cell proliferation and metastasis in EC. Subsequently, Western blotting was used to detect the expression level of PI3K/AKT in EC. Besides, we used the tumor formation assay in vivo to examine the ability of LINP1 in tumor formation in vivo. RESULTS: LINP1 was proved to be up-regulated in EC cell lines and tissues by qRT-PCR assay. CCK-8 assay and colony formation assay were conducted and the results indicated that LINP1 over-expression can promote cell proliferation in EC in vitro. The data of transwell and Matrigel assays indicated that up-regulated LINP1 can facilitate cell migration and invasion. The results of Western blotting validated that LINP1 can activate PI3K/AKT signaling. Besides, the tumor formation assay verified that LINP1 can promote tumor formation in vivo. CONCLUSIONS: Our research validated that LINP1 served as an oncogenic role in EC progression. The PI3K/AKT signaling pathway might be the underlying mechanism of EC progression. We hope our study can provide novel treatment targets and biomarkers in EC development and progression.

Nanoparticle-based delivery of an anti-proliferative metal chelator to tumor cells.

This paper describes the preparation and characterization of polymeric nanoparticles loaded with a potent anti-tumor metal chelator, Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) for delivery to cancer cells. Metal chelators have been increasingly studied for their anti-cancer properties that rely on the high demand of neoplastic cells for iron. Dp44mT has previously shown great antiproliferative characteristics in several cancers including breast cancer and melanoma. To further expand the application of this highly cytotoxic agent for cancer treatment and to enable its specific delivery to malignant cells, here we apply nano-scale particles (NPs) of biodegradable poly(lactic-co-glycolide) (PLGA) for encapsulation of Dp44mT and evaluate its effectiveness in vitro. The results demonstrated that Dp44mT was efficiently encapsulated in PLGA particles. Resulting NPs were uniform in size and shape and had good colloidal stability. Moreover, Dp44mT encapsulation in PLGA enhanced the water solubility of this agent. Lastly, the present formulation showed high level of cytotoxicity in glioma cells. Together, these results show the potential of PLGA NPs as a nano-carrier for Dp44mT with no apparent impact on the anti-tumor activity of this compound.

The prognostic role of tertiary Gleason pattern 5 in a contemporary grading system for prostate cancer.

BACKGROUND: Recently, a new prostate cancer (PC) grading system has been introduced, where Gleason score (GS) 7 (3+4) and GS 7 (4+3) are categorized into two separate groups. However, GS 7 with tertiary Gleason pattern 5 (TGP5) was not incorporated in the new grading system. In the present study, we validated the prognostic role of TGP5 in the new classification. METHODS: We retrospectively reviewed the records of 1396 patients with localized GS 6-8 PC (pT2-3N0M0) who underwent radical prostatectomy at our institution between 2005 and 2014. After excluding patients who received neoadjuvant or adjuvant therapy, or had incomplete pathological or follow-up data, 1229 patients were included in the final analysis. The Kaplan-Meier method was used to estimate and compare the probabilities of biochemical recurrence (BCR). Cox regression models were used to investigate associations between variables and the risk of BCR. RESULTS: Of 732 GS 7 patients, 75 (10.2%) had a TGP5. The BCR-free survival rate for men with TGP5 was significantly worse than for those without TGP5 (P<0.001). In multivariate Cox regression analyses for GS 7 PC, TGP5 was a significant predictor of BCR (hazard ratio 1.750, P=0.027). When the total cohort was stratified into four grade groups according to the new classification, group 2 with TGP5 had a BCR risk comparable to group 3, and group 3 with TGP5 behaved like group 4. CONCLUSIONS: Our study shows that TGP5 increased the BCR risk after RP in GS 7 PC. Moreover, we demonstrated that the presence of a TGP5 in GS 7 upgraded the BCR risk to one comparable with the next higher category under the new classification. These findings support incorporating TGP5 into GS 7 to aid with future risk assessment and follow-up scheduling for PC.

Prognostic impact of preoperative neutrophil-to-lymphocyte ratio after radical prostatectomy in localized prostate cancer.

BACKGROUND: Neutrophil-to-lymphocyte ratio (NLR) has a prognostic value in patients with metastatic castration-resistant prostate cancer receiving systemic therapy. However, the prognostic significance of NLR was never previously evaluated in patients who underwent radical prostatectomy (RP) for prostate cancer. In the present study, we investigated the influence of NLR on survival after a RP for prostate cancer. METHODS: We retrospectively reviewed clinical data of 2301 patients with prostate cancer who underwent RP at our institution between 2000 and 2010. Among these patients, we considered only patients who had a preoperative complete blood count with differential result available. Patients who received neoadjuvant or postoperative adjuvant treatment (radiation, androgen deprivation therapy or both) and those without adequate medical record were excluded. A Kaplan-Meier analysis was performed to analyze biochemical recurrence-free survival (BCRFS), overall survival (OS) and prostate cancer-specific survival (CSS). Univariate and multivariate Cox regression models were used for each end point. RESULTS: In total, 2067 patients were evaluated; median follow-up time was 78 months (interquartile range (IQR) 65-96), median age at RP was 66 years (IQR 61-70) and median preoperative NLR was 1.76 (IQR 1.35-2.40). A Kaplan-Meier analysis showed a significant association between high NLR (⩾1.76) and decreased CSS (P=0.005) and OS (P=0.003) but not with BCRFS (P=0.223). In the univariate and multivariate regression analyses, a high NLR was a significant predictor of CSS (hazard ratio (HR) 2.012, 95% confidence interval (CI) 1.222-3.310, P=0.006) and OS (HR 1.650, 95% CI 1.127-2.416, P=0.010). CONCLUSIONS: This study shows that in patients with prostate cancer preoperative NLR is an independent prognostic factor for OS and CSS after a RP and suggests that a preoperative hematologic workup should be considered in the risk assessment of these patients.

Successful withdrawal of antiviral treatment in kidney transplant recipients with chronic hepatitis B viral infection.

BACKGROUND: The optimal duration of antiviral therapy for kidney transplant recipients (KTR) with chronic hepatitis B virus (HBV) infection remains unclear. We reported the long-term outcomes after withdrawal of antiviral agent in KTR with chronic HBV infection. METHODS: We retrospectively investigated the hepatitis B surface antigen (HBsAg)-positive KTR with antiviral agents between January 2002 and January 2012. Antiviral treatments were withdrawn in patients who met all of the following 7 criteria: (i) no clinical and histologic evidence of cirrhosis, (ii) normal liver biochemistry, (iii) negative for both HBV DNA and hepatitis B envelope antigen (HBeAg), (iv) no resistance to antiviral agent, (v) antiviral therapy > 9 months, (vi) maintenance dosage of immunosuppressant for > 3 months, and (vii) no history of acute rejection during recent 6 months. All patients were followed regularly at approximately 3-6 months for liver enzyme, viral markers, and HBV DNA level after antiviral withdrawal. RESULTS: Among a total of 445 KTR, 14 HBsAg-positive patients were included in this study. Antiviral agents were used, with lamivudine in 11 patients, and with adefovir, entecavir, and telbivudine in 3 patients, respectively. Discontinuation of antiviral agent was attempted in 6 (42.9%) of 14 patients who satisfied the criteria. The median duration of antiviral therapy before withdrawal was 14.3 months (range, 9-24 months). Four (66.7%) of 6 patients were successfully withdrawn and remained negative for HBV DNA for a median 60.5 months (range, 47-82 months). The baseline HBV DNA level was not related to maintenance of remission after withdrawal. Two reactivated patients resumed antiviral treatment immediately, with subsequent normalization of HBV DNA. During the follow-up, 1 patient developed hepatocellular carcinoma; however, no patient death or graft failure was reported for all HBsAg-positive KTR. CONCLUSIONS: Antiviral therapy can be discontinued successfully and safely in selected KTR with chronic HBV infection, after complete suppression of HBV and sufficient duration of antiviral therapy.

Stronger proteasomal inhibition and higher CHOP induction are responsible for more effective induction of paraptosis by dimethoxycurcumin than curcumin.

Although curcumin suppresses the growth of a variety of cancer cells, its poor absorption and low systemic bioavailability have limited its translation into clinics as an anticancer agent. In this study, we show that dimethoxycurcumin (DMC), a methylated, more stable analog of curcumin, is significantly more potent than curcumin in inducing cell death and reducing the clonogenicity of malignant breast cancer cells. Furthermore, DMC reduces the tumor growth of xenografted MDA-MB 435S cells more strongly than curcumin. We found that DMC induces paraptosis accompanied by excessive dilation of mitochondria and the endoplasmic reticulum (ER); this is similar to curcumin, but a much lower concentration of DMC is required to induce this process. DMC inhibits the proteasomal activity more strongly than curcumin, possibly causing severe ER stress and contributing to the observed dilation. DMC treatment upregulates the protein levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and Noxa, and the small interfering RNA-mediated suppression of CHOP, but not Noxa, markedly attenuates DMC-induced ER dilation and cell death. Interestingly, DMC does not affect the viability, proteasomal activity or CHOP protein levels of human mammary epithelial cells, suggesting that DMC effectively induces paraptosis selectively in breast cancer cells, while sparing normal cells. Taken together, these results suggest that DMC triggers a stronger proteasome inhibition and higher induction of CHOP compared with curcumin, giving it more potent anticancer effects on malignant breast cancer cells.

The differential profiles of withdrawal symptoms induced by morphine and beta-endorphin administered intracerebroventricularly in mice.

In the present study, withdrawal symptoms induced by morphine or ß-endorphin administered intracerebroventricularly (i.c.v.) were compared in ICR mice. Naloxone (10mg/kg) was post-treated intraperitoneally (i.p.) 3h after either a single or repeated (1 time/day for 3 days) i.c.v. injections with opioids. Withdrawal symptoms such as jumping frequency, diarrhea, weight loss, rearing, penile licking and paw tremor were observed for 30 min immediately after naloxone treatment. Withdrawal symptoms (jumping, diarrhea, weight loss, rearing, penile licking and paw tremor) observed in the group treated with morphine was persistently increased during 3 days. On the other hand, withdrawal symptoms such as diarrhea, weight loss and rearing in ß-endorphin-treated group were increased after a single injection with ß-endorphin, but gradually decreased after the repeated injection. Furthermore, no jumping behavior, penile licking and paw tremor in ß-endorphin-treated group were observed throughout the whole period of time. In addition, the hypothalamic changes of several signal molecules such as pERK, pCaMK-IIα, c-FOS and pCREB expression were observed during the presence or absence of withdrawal responses induced by morphine or ß-endorphin administered once or repeatedly. Both hypothalamic pCaMK-IIα and c-FOS expressions were increased by naloxone treatment in acutely administered morphine group, whereas only pCaMK-IIα expression was elevated by naloxone treatment in repeatedly administered morphine group. In contrast with the findings in morphine-treated group, only pCaMK-IIα expression was decreased by naloxone treatment in repeatedly administered ß-endorphin group. Our results suggest that profiles of the withdrawal symptoms induced by morphine and ß-endorphin administered supraspinally appear to be differentially regulated. The pCaMK-IIα and the c-FOS protein expression may play important roles for the regulation of naloxone-precipitated withdrawal symptoms such as jumping, diarrhea, weight loss, rearing, penile licking and paw tremor induced by morphine-treated group, whereas the phosphorylation of hypothalamic pCaMK-IIα appears to be involved only in the regulation of naloxone-precipitated withdrawal symptoms such as diarrhea, weight loss and rearing in ß-endorphin-treated group.

The relationship between mast cell density and tumour grade in transitional cell carcinoma of the bladder.

This study investigated the relationship between mast cell numbers and the grade of bladder transitional cell carcinoma (TCC). Bladder TCC biopsies were obtained via transurethral resection and 45 stage T1 specimens were included in the study. Specimens were sorted into two groups, low grade (grade I) and high grade (grades II and III). Samples were stained using haematoxylin and eosin, toluidine blue and immunohistochemical staining for tryptase. Mast cells were examined by light microscopy and cell density was recorded. Mast cell density was significantly higher in high-grade TCC than low-grade TCC. There was also a significant relationship between the number of mast cells identified using toluidine blue staining or immunohistochemical staining for tryptase. Detailed studies of mast cell function will enable the development of more effective antitumour therapies via mast cell manipulation.

Treatment guidelines for isolated dissection of the superior mesenteric artery based on follow-up CT findings.

OBJECTIVES: The treatment guidelines for isolated superior mesenteric artery dissection (SMAD) are not well established. The purpose of this study was to report a single-centre series of SMAD and propose treatment guidelines. MATERIALS AND METHODS: Between November 2004 and December 2009, 30 patients were diagnosed with SMAD. We retrospectively reviewed their medical records. RESULTS: The subjects included 26 men and four women, with a mean age of 55.1 years. The chief complaint was abdominal pain in 17 patients, whereas 13 patients were asymptomatic. The mean follow-up was 38.3 months. The radiographic findings included intimal flap with a false lumen in 20 patients and intramural haematoma in 10 patients. The treatments included observation in 18 patients, anticoagulation in five patients, stenting in six patients and surgery in one patient. During follow-up (mean 15.6 months), there was no change in the computed tomography scans of seven patients, improvement was observed in four patients and complete resolution was observed in four patients. All patients, including the symptomatic patients, remained asymptomatic during follow-up. CONCLUSIONS: Most patients with SMAD can be successfully managed with conservative treatment. Surgical treatment or percutaneous intervention can be reserved for patients with severe mesenteric ischaemia and those for whom the initial conservative treatment fails.

The TSC1 and TSC2 tumor suppressors are required for proper ER stress response and protect cells from ER stress-induced apoptosis.

Tuberous sclerosis complex (TSC)1 and TSC2 are tumor suppressors that inhibit cell growth and mutation of either gene causes benign tumors in multiple tissues. The TSC1 and TSC2 gene products form a functional complex that has GTPase-activating protein (GAP) activity toward Ras homolog enriched in brain (Rheb) to inhibit mammalian target of rapamycin complex 1 (mTORC1), which is constitutively activated in TSC mutant tumors. We found that cells with mutation in either TSC1 or TSC2 are hypersensitive to endoplasmic reticulum (ER) stress and undergo apoptosis. Although the TSC mutant cells show elevated eIF2α phosphorylation, an early ER stress response marker, at both basal and induced conditions, induction of other ER stress response markers, including ATF4, ATF6 and C/EBP homologous protein (CHOP), is severely compromised. The defects in ER stress response are restored by raptor knockdown but not by rapamycin treatment in the TSC mutant cells, indicating that a rapamycin-insensitive mTORC function is responsible for the defects in ER stress response. Consistently, activation of Rheb sensitizes cells to ER stress. Our data show an important role of TSC1/TSC2 and Rheb in unfolded protein response and cell survival. We speculate that an important physiological function of the TSC1/2 tumor suppressors is to protect cells from harmful conditions. These observations indicate a potential therapeutic application of using ER stress agents to selectively kill TSC1 or TSC2 mutant cells for TSC treatment.

Mesenchymal stem cells overexpressing interleukin-10 attenuate collagen-induced arthritis in mice.

Mesenchymal stem cells (MSCs) have the inherent ability to migrate to multiple organs and to exert immunosuppressive activity. The aim of this study was to investigate the anti-arthritogenic effects of interleukin (IL)-10-transduced MSCs (IL-10-MSC) on the development of inflammatory arthritis. DBA/1 mice were immunized with type II collagen (CII) to induce inflammatory arthritis and then injected weekly three times with IL-10-MSCs 21 days after primary immunization. Control mice received vehicle or MSCs alone. Serum anti-CII antibody and T cell response to CII were determined. The results showed that cultured IL-10-MSCs were able to secrete high amounts of IL-10 in vitro. Injection of IL-10-MSCs decreased the severity of arthritis significantly. However, there was no difference in arthritis severity between mice treated with MSC and vehicle alone. Anti-CII antibody titres in the sera and T cell proliferative response to CII in lymph node cells were decreased significantly in mice treated with IL-10-MSCs compared with vehicle-treated mice. Serum IL-6 level was also decreased by the administration of IL-10-MSCs. In contrast, spleen cells of IL-10-MSC-treated mice produced higher amounts of IL-4 than those of control mice. Interestingly, although not as potent as IL-10-MSCs, injection of naive MSCs alone decreased serum levels of IL-6 and anti-CII antibody, while increasing IL-4 production from cultured splenic cells. Taken together, systemic administration of genetically modified MSCs overexpressing IL-10 inhibits experimental arthritis not only by suppressing autoimmune response to CII but also by regulating cytokine production, and thus would be a new strategy for treating rheumatoid arthritis.

Zinc inhibition of caspase-3 activation does not protect HeLa cells from apoptotic cell death.

Zinc is proposed to be antiapoptotic for it has been shown to inhibit late events of apoptotic pathways such as Ca(2+)/Mg(2+)-dependent endonuclease cleavage of chromatin DNA, poly-ADP ribose polymerase cleavage, and caspase-3 activity. Because caspase-3 is a critical executioner caspase in apoptosis, this study was undertaken to examine specifically a correlation between zinc inhibition of caspase-3 activation and apoptosis in HeLa cells. Cultured HeLa cells were exposed to 100 microM ZnCl(2) for 1 h prior to 12 h treatment with 1.0 microM doxorubicin (DOX), an important anticancer agent that causes apoptosis in a wide variety of tumor cells. Western blot analysis of HeLa cells treated with DOX for 12 h revealed that DOX caused proteolytic activation of caspase-3 and zinc inhibited this activation. Interestingly, zinc did not inhibit DOX-induced apoptosis as measured by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Furthermore, a microculture tetrazolium assay confirmed that cell death occurred in the presence of zinc. These results demonstrate that zinc specifically inhibits DOX-induced activation of caspase-3 in HeLa cells, but does not suppress DOX-induced apoptosis or otherwise cell death, thus suggesting DOX-induced caspase-3 activation may not play a major role in overall cell death and/or non-caspase-3 pathways are involved in DOX-induced apoptosis in HeLa cells.

Ethanol-induced apoptosis in mouse liver: Fas- and cytochrome c-mediated caspase-3 activation pathway.

Hepatic apoptosis has been shown to occur in both experimental and clinical alcoholic liver disease, but the signaling pathway remains unknown. This study was undertaken to examine specifically the involvement of the upstream signals, Fas and cytochrome c, in alcohol-induced caspase-3 activation and apoptosis in the liver. Male FVB mice were administrated intragastrically a single dose of alcohol at 6 g/kg, which has been shown to represent binge drinking in humans. Hepatic apoptosis was detected by a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Active form of caspase-3 was identified by immunoperoxidase staining and confirmed by immunogold labeling and was found to be in the cytosol and nucleus. Enzymic assay further confirmed caspase-3 activation and nucleus localization. Systemic administration of caspase-3 inhibitor, Ac-DEVD-FMK, inhibited caspase-3 activity and abrogated apoptosis. Elevation of cytosolic cytochrome c was found by immunoperoxidase staining, immunogold labeling, and Western blot. Increased Fas ligand expression was detected by immunoperoxidase staining. Intravenous administration of a neutralizing Fas ligand monoclonal antibody resulted in suppression of caspase-3 activation and attenuation of apoptosis, but did not inhibit mitochondrial cytochrome c release. The results thus demonstrate that Fas/Fas ligand system-mediated caspase-3 activation plays a central role in the ethanol-induced hepatic apoptosis.

Metallothionein inhibits doxorubicin-induced mitochondrial cytochrome c release and caspase-3 activation in cardiomyocytes.

Previous studies using transgenic mice in which metallothionein (MT) was overexpressed only in the heart have demonstrated that MT protects from oxidative cardiac injury induced by doxorubicin (DOX), an important anticancer agent. MT cardioprotection is associated with its antiapoptotic effect. The present study was undertaken to test the hypothesis that MT suppresses DOX-induced apoptosis through inhibition of mitochondrial cytochrome c release and caspase-3 activation. Primary cultures of cardiomyocytes isolated from the hearts of transgenic neonatal mice and nontransgenic controls were treated with DOX at a clinically relevant concentration (1.0 microM) for varying time periods. Apoptosis was detected in nontransgenic cardiomyocyte cultures by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Annexin V-fluorescein isothiocyanate binding. This apoptotic effect was significantly suppressed in the MT-overexpressing transgenic cardiomyocytes. Western blot analysis revealed that DOX caused mitochondrial cytochrome c release. Furthermore, caspase-3 activation was observed. The activation of this apoptotic pathway by DOX was dramatically inhibited in the MT-overexpressing cardiomyocytes. To elucidate the role of reactive oxygen species (ROS) in the activation of the cytochrome c-mediated caspase-3 activation pathway, the intracellular levels of ROS and their localization were detected by fluorescent confocal microscopy. Mitochondrial ROS concentrations were dramatically elevated by DOX in nontransgenic cardiomyocytes. This elevation was completely inhibited almost in the MT-overexpressing cardiomyocytes. Thus, these results demonstrate that MT suppresses DOX-induced apoptosis in cardiomyocytes through, at least in part, inhibition of the cytochrome c-mediated apoptotic pathway.

Attenuation of doxorubicin chronic toxicity in metallothionein-overexpressing transgenic mouse heart.

Previous studies have shown that cardiac-specific metallothionein (MT)-overexpressing transgenic mice are highly resistant to acute cardiotoxicity induced by doxorubicin (DOX), a most effective anticancer agent. However, cumulative dose-dependent chronic cardiotoxicity attributable to long-term administration of DOX is a significant clinical problem. Because MT is a potent antioxidant and oxidative stress is critically involved in DOX-induced heart injury, the present study was undertaken to test the hypothesis that MT also provides protection against DOX chronic cardiotoxicity. Transgenic mice containing high levels of cardiac MT and nontransgenic controls were treated with a cumulative dose of 40 mg/kg of DOX in 10 equal i.v. injections over a period of 7 weeks. Three weeks after the last injection, the mice were killed for an analysis of cardiotoxicity. As compared with nontransgenic controls, DOX-induced cardiac hypertrophy was significantly inhibited in the transgenic mice. Light microscopic examination revealed that DOX-induced myocardial morphological changes were markedly suppressed or almost eliminated in the transgenic mice. Under electron microscopy, extensive sarcoplasmic vacuolization and severe disruption of mitochondrial fine structure were observed in nontransgenic cardiomyocytes, but almost no sarcoplasmic vacuolization was observed, and the mitochondrial structural changes were almost completely prevented in the transgenic cardiomyocytes. The results thus indicate that MT elevation is a highly effective approach to prevent chronic cardiomyopathy attributable to DOX. This study also suggests that oxidative stress is critically involved in the DOX-induced chronic cardiotoxicity.

Molecular and cellular mechanisms of cardiotoxicity.

Cardiotoxicity resulting from detrimental environmental insults has been recognized for a long time. However, extensive studies of the mechanisms involved had not been undertaken until recent years. Advances in molecular biology provide powerful tools and make such studies possible. We are gathering information about cellular events, signaling pathways, and molecular mechanisms of myocardial toxicologic responses to environmental toxicants and pollutants. Severe acute toxic insults cause cardiac cell death instantly. In the early response to mild environmental stimuli, biochemical changes such as alterations in calcium homeostasis occur. These may lead to cardiac arrhythmia, which most often is reversible. Prolonged stimuli activate transcription factors such as activator protein-1 through elevation of intracellular calcium and the subsequent activation of calcineurin. Upregulation by activated transcription factors of hypertrophic genes results in heart hypertrophy, which is a short-term adaptive response to detrimental factors. However, further development of hypertrophy will lead to severe and irreversible cardiomyopathy, and eventually heart failure. From cardiac hypertrophy to heart failure, myocardial cells undergo extensive biochemical and molecular changes. Cardiac hypertrophy causes tissue hypoperfusion, which activates compensatory mechanisms such as production of angiotensin II and norepinephrine. Both further stimulate cardiac hypertrophy and, importantly, activate counterregulatory mechanisms including overexpression of atrial natriuretic peptide and b-type natriuretic peptide, and production of cytokines such as tumor necrosis factor-alpha. This counterregulation leads to myocardial remodeling as well as cell death through apoptosis and necrosis. Cell death through activation of mitochondrial factors and other pathways constitutes an important cellular mechanism of heart failure. Our current knowledge of cardiotoxicity is limited. Further extensive studies are warranted for a comprehensive understanding of this field.