CD44 Receptor-Mediated/Reactive Oxygen Species-Sensitive Delivery of Nanophotosensitizers against Cervical Cancer Cells.

Stimulus-sensitive, nanomedicine-based photosensitizer delivery has an opportunity to target tumor tissues since oxidative stress and the expression of molecular proteins, such as CD44 receptors, are elevated in the tumor microenvironment. The aim of this study is to investigate the CD44 receptor- and reactive oxygen species (ROS)-sensitive delivery of nanophotosensitizers of chlorin e6 (Ce6)-conjugated hyaluronic acid (HA) against HeLa human cervical cancer cells. For the synthesis of nanophotosensitizers, thioketal diamine was conjugated with the carboxyl group in HA and then the amine end group of HA-thioketal amine conjugates was conjugated again with Ce6 (Abbreviated as HAthCe6). The HAthCe6 nanophotosensitizers were of small diameter, with sizes less than 200. Their morphology was round-shaped in the observations using a transmission electron microscope (TEM). The HAthCe6 nanophotosensitizers responded to oxidative stress-induced changes in size distribution when H2O2 was added to the nanophotosensitizer aqueous solution, i.e., their monomodal distribution pattern at 0 mM H2O2 was changed to dual- and/or multi-modal distribution patterns at higher concentrations of H2O2. Furthermore, the oxidative stress induced by the H2O2 addition contributed to the disintegration of HAthCe6 nanophotosensitizers in morphology, and this phenomenon accelerated the release rate of Ce6 from nanophotosensitizers. In a cell culture study using HeLa cells, nanophotosensitizers increased Ce6 uptake ratio, ROS generation and PDT efficacy compared to free Ce6. Since HA specifically bonds with the CD44 receptor of cancer cells, the pretreatment of free HA against HeLa cells decreased the Ce6 uptake ratio, ROS generation and PDT efficacy of HAthCe6 nanophotosensitizers. These results indicated that intracellular delivery of HAthCe6 nanophotosensitizers can be controlled by the CD44 receptor-mediated pathway. Furthermore, these phenomena induced CD44 receptor-controllable ROS generation and PDT efficacy by HAthCe6 nanophotosensitizers. During in vivo tumor imaging using HeLa cells, nanophotosensitizer administration showed that the fluorescence intensity of tumor tissues was relatively higher than that of other organs. When free HA was pretreated, the fluorescence intensity of tumor tissue was relatively lower than those of other organs, indicating that HAthCe6 nanophotosensitizers have CD44 receptor sensitivity and that they can be delivered by receptor-specific manner. We suggest that HAthCe6 nanophotosensitizers are promising candidates for PDT in cervical cancer.

Redox-Sensitive and Folate-Receptor-Mediated Targeting of Cervical Cancer Cells for Photodynamic Therapy Using Nanophotosensitizers Composed of Chlorin e6-Conjugated ß-Cyclodextrin via Diselenide Linkage.

The aim of this study was to fabricate a reactive oxygen species (ROS)-sensitive and folate-receptor-targeted nanophotosensitizer for the efficient photodynamic therapy (PDT) of cervical carcinoma cells. Chlorin e6 (Ce6) as a model photosensitizer was conjugated with succinyl ß-cyclodextrin via selenocystamine linkages. Folic acid (FA)-poly(ethylene glycol) (PEG) (FA-PEG) conjugates were attached to these conjugates and then FA-PEG-succinyl ß-cyclodextrin-selenocystamine-Ce6 (FAPEGbCDseseCe6) conjugates were synthesized. Nanophotosensitizers of FaPEGbCDseseCe6 conjugates were fabricated using dialysis membrane. Nanophotosensitizers showed spherical shapes with small particle sizes. They were disintegrated in the presence of hydrogen peroxide (H2O2) and particle size distribution changed from monomodal distribution pattern to multimodal pattern. The fluorescence intensity and Ce6 release rate also increased due to the increase in H2O2 concentration, indicating that the nanophotosensitizers displayed ROS sensitivity. The Ce6 uptake ratio, ROS generation and cell cytotoxicity of the nanophotosensitizers were significantly higher than those of the Ce6 itself against HeLa cells in vitro. Furthermore, the nanophotosensitizers showed folate-receptor-specific delivery capacity and phototoxicity. The intracellular delivery of nanophotosensitizers was inhibited by folate receptor blocking, indicating that they have folate-receptor specificity in vitro and in vivo. Nanophotosensitizers showed higher efficiency in inhibition of tumor growth of HeLa cells in vivo compared to Ce6 alone. These results show that nanophotosensitizers of FaPEGbCDseseCe6 conjugates are promising candidates as PDT of cervical cancer.

A Case of "Sinking Skin Flap Syndrome" in Vegetative State Patient.

Sinking skin flap syndrome is defined by a series of neurological symptoms with skin depression at the site of cranial defect. We experienced neurological improvement in a patient with markedly sunken craniectomy site after ventriculoperitoneal shunt (V-P shunt) clamping operation. A 17-year old female patient was in vegetative state and spastic quadriplegia after traumatic brain injury. She was suffered from frequent vomiting. To evaluate central nervous system problem we checked brain computed tomography which showed that right frontotemporoparietal craniectomy area was markedly sunken and midline was shifting to the left. After V-P shunt clamping operation, craniectomy site was elevated and midline shifting was improved. Vomiting was disappeared. Coma Recovery Scale-revised (CRS-R) score was improved from 3 to 6.

Efficacy of auditory evoked potential follow-up in viral meningitis of infants.

PURPOSE: To examine the rate of occurrence of hearing impairments among the infants who had recovered from viral meningitis under 1 year of age through auditory evoked potential (AEP) test and to investigate the efficacy of the follow-up AEP test in viral meningitis infants. METHODS: Two hundred twenty infants (440 ears) were examined through AEP test once, and 47 (94 ears) of them went back for a second examination and were diagnosed with viral meningitis. The first AEP tests were compared with the second results in 47 infants. I latency, V latency, I-III interpeak latency (IPL), and III-V IPL were checked. RESULTS: In the first AEP test conducted on 440 ears, the average values of I and V latency and I-III IPL were delayed as compared with normal values. The second AEP results were conducted on 47 infants 92.36 days after the first exam. I latency and V latency of second exam were improved significantly (p < 0.05), but I-III and III-V IPL showed no significant changes. Two hearing impaired patients (4 ears) were confirmed through chart reviews. CONCLUSION: The AEP test is a helpful study for early detection of hearing problem. However, in this study, AEP test was too sensitive in acute period, and later, the incidence rate of hearing impairment was relatively low. Therefore, age of onset, severity of neurologic symptom, and clinical examination must be considered before the AEP test.

Expression of steroidogenic enzymes in human placenta according to the gestational age.

Female sex steroid hormones, including estradiol (E2) and progesterone (P4), serve significant physiological roles in pregnancy. In particular, E2 and P4 influence placenta formation, maintain pregnancy and stimulate milk production. These hormones are produced by ovaries, adrenal glands and the placenta, of which the latter is a major endocrine organ during pregnancy. However, the mechanism of hormone production during pregnancy remains unclear. In the present study, the regulation of steroid hormones and steroidogenic enzymes was examined in human placenta according to gestational age. In human placental tissues, expression levels of steroidogenic enzymes were determined with reverse transcription­quantitative polymerase chain reaction and western blotting. The mRNA and protein expression of CYP17A1, HSD17B3 and CYP19A1, which are associated with the synthesis of dehydroepiandrosterone (DHEA) and E2, was elevated at different gestational ages in human placenta. In addition, to evaluate the correlation between serum and placental­produced hormones, steroid hormone levels, including pregnenolone (PG), DHEA, P4, testosterone (T) and E2, were examined in serum and placenta. Serum and placenta expression of DHEA and E2 increased with gestational age, whereas T and P4 were differently regulated in placenta and serum. To confirm the mechanism of steroidogenesis in vitro, placental BeWo cells were treated with E2 and P4, which are the most important hormones during pregnancy. The mRNA and protein expression of steroidogenic enzymes was significantly altered by E2 in vitro. These results demonstrated that concentration of steroid hormones was differently regulated by steroidogenic enzymes in the placenta depending on the type of the hormones, which may be critical to maintain pregnancy.

The regulation of oxytocin and oxytocin receptor in human placenta according to gestational age.

Oxytocin (OXT) is a peptide hormone that plays a central role in the regulation of parturition and lactation. OXT signaling is mediated by OXT receptor (OXTR), which shows species- and tissue-specific expressions and gene regulation. In the present study, we examined the synthesis of OXT and OXTR in human placenta tissue according to gestational age. A total of 48 placentas were divided into early preterm, late preterm and term groups depending on gestational age, and expression of OXT and OXTR was evaluated. First, OXT and OXTR mRNA and protein were detected in normal placenta tissue via Q-PCR, Dot-blot and Western blot assay. Both OXT and OXTR levels in normal placenta increased gradually in the late stage of pregnancy, suggesting that local OXT may play a critical role in the function of the placenta. To determine the regulatory mechanism of OXT, placental BeWo cells were administrated estrogen (E2) or progesterone (P4), and expression of OXT and OXTR was tested. The mRNA and protein levels of OXT and OXTR were upregulated by E2 but blocked by co-treatment with P4 In order to confirm the estrogen receptor (ESR)-mediated signaling, we administrated ESR antagonists together with E2 to BeWo cells. As a result, both OXT and OXTR were significantly altered by ESR1 antagonist (MPP) while moderately regulated by ESR2 antagonist (PHTPP). These results suggest that OXT and OXTR are controlled mainly by E2 in the placenta via ESR1 and thus may play physiological functions in the human placenta during the late stage of pregnancy.

Pregnenolone sulfate regulates prolactin production in the rat pituitary.

Pregnenolone sulfate (PS) is a neuroactive steroid hormone produced in the brain. In this study, the effects of PS on synthesis and secretion of rat pituitary prolactin (PRL) were examined. To accomplish this, GH3 rat pituitary adenoma cells were treated with PS, which showed significantly increased mRNA and protein levels of PRL compared with the control. The mechanism of action responsible for the effects of PS on PRL synthesis and secretion was investigated by pretreating cells with inhibitors of traditional PRL- or the PS-related signaling pathway. PS-stimulated PRL transcription was significantly reduced by inhibitors of PKA, PKC and MAPK, but unchanged by GABAAR and NMDAR inhibitors. Western blotting analysis revealed that the total ERK1/2 level was upregulated in a time-dependent manner following PS treatment. An approximate 10% increase in GH3 cell proliferation was also observed in response to PS relative to the control. In the animal study, levels of PRL in the pituitary and in serum were elevated by PS. PS-stimulated PRL synthesis was also found to be associated with decreased expression of PRL target genes such as GNRH1, FSHB and LHB. These findings show that PS upregulates PRL synthesis and secretion in vivo and in vitro via MAPK signaling, suggesting that it has the potential for use as a therapeutic hormone to treat PRL-related disorders such as hypoprolactinemia and low milk supply.

Effects of alpha-tocopherol on cadmium-induced toxicity in rat testis and spermatogenesis.

Cadmium is known to exert toxic effects on multiple organs, including the testes. To determine if alpha-tocopherol, an antioxidant, could protect testicular tissues and spermatogenesis from the toxic effects of cadmium, six-week old male Sprague-Dawley rats were randomized to receive cadmium at doses of 0 (control), 1, 2, 4 or 8 mg/kg by the intraperitoneal route (Group A) or alpha-tocopherol for 5 days before being challenged with cadmium (Group B) in an identical dose-dependent manner. When both groups received cadmium at 1 mg/kg, there were no changes in testicular histology relative to controls. When Group A received cadmium at 2 mg/kg, undifferentiated spermatids and dead Sertoli cells increased in the seminiferous tubules while interstitial cells decreased and inflammatory cells increased in the interstitial tissues. On flow cytometric analysis, the numbers of elongated spermatids (M1) and round spermatids (M2) decreased while 2c stage cells (M3, diploid) increased. In contrast, when Group B received cadmium at 2 mg/kg, the histological insults were reduced and the distribution of the germ cell population remained comparable to controls. However, alpha-tocopherol had no protective effects with higher cadmium doses of 4 and 8 mg/kg. These findings indicate that alpha-tocopherol treatment can protect testicular tissue and preserve spermatogenesis from the detrimental effects of cadmium but its effectiveness is dependent on the dose of cadmium exposed.