Correlation between plasma lycopene levels in patients with laryngeal carcinoma and postoperative adverse complications of chemoradiotherapy and nutritional risks.

OBJECTIVE: In this study, we analyzed the correlation between the preoperative plasma lycopene levels, postoperative adverse complications of chemoradiotherapy, and nutritional risk scores in patients with laryngeal carcinoma. METHODS: A total of 114 patients with laryngeal carcinoma and 114 healthy respondents were enrolled in this study. The patients with laryngeal carcinoma were divided into two groups: 62 patients with laryngeal carcinoma, with an NRS2002 score higher than 3 points and whose diet contained lycopene, were enrolled in the observation group, and 52 patients with laryngeal carcinoma during the corresponding time period, whose diet did not contain lycopene, were enrolled in the reference group. The immune indexes (CD4 + , CD8 + , IGA, IGM, IGG), nutritional indexes (albumin, prealbumin, transferrin), and postoperative adverse complications of chemo-radiotherapy in the two groups were recorded. RESULTS: The lycopene levels were lower in patients with advanced tumor stage (III and IV). The diagnosis threshold of the plasma lycopene level for laryngeal carcinoma was 0.503 µmol/L. The area under the curve for plasma lycopene levels in cancer diagnosis was 0.96, with a clinical specificity of 0.943 and a sensitivity of 0.859. There was a significant negative correlation between the plasma lycopene levels and Nutrition Risk Screening (NRS) 2002 score (R2 = - 0.523, P < 0.001), which was related to the increase in NRS-2002 scores and nutritional hazards in patients with laryngeal carcinoma. The observation group showed a significant increase in nutritional and immune indices, as compared to the reference group, as well as a lower incidence of severe and serious adverse reactions to chemo-radiotherapy. Lycopene supplementation, tumor stage, NRS-2002 scores, nutritional and immune indices were all significant predictors of postoperative severe and serious adverse complications of chemoradiotherapy. CONCLUSION: Progression of laryngeal carcinoma and severity of the side effects of the adverse complications of chemo-radiotherapy are related to the levels of lycopene.

Platelet-Rich Plasma Gel-Loaded Collagen/Chitosan Composite Film Accelerated Rat Sciatic Nerve Injury Repair.

Peripheral nerve injury (PNI) is a common clinical disease caused by severe limb trauma, congenital malformations, and tumor resection, which may lead to significant functional impairment and permanent disability. Nerve conduit as a method for treating peripheral nerve injury shows good application prospects. In this work, the COL/CS composite films with different mass ratios of 1:0, 1:1, and 1:3 were fabricated by combining physical doping. Physicochemical characterization results showed that the COL/CS composite films possessed good swelling properties, ideal mechanical properties, degradability and suitable hydrophilicity, which could meet the requirements of nerve tissue engineering. In vitro cell experiments showed that the loading of platelet-rich plasma (PRP) gel on the surface of COL/CS composite films could significantly improve the biocompatibility of films and promote the proliferation of Schwann cells. In addition, a rat model of sciatic nerve defect was constructed to evaluate the effect of COL/CS composite films on peripheral nerve repair and the results showed that COL/CS composite films loaded with PRP gel could promote nerve regeneration and functional recovery in rats with sciatic nerve injury, indicating that the combination of PRP gel with the COL/CS composite film would be a potential approach for the treatment of peripheral nerve injury.

Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations.

Anaplastic thyroid cancer is an extremely lethal malignancy without reliable treatment. BRAFV600E point mutation is common in ATCs, which leads to MAPK signaling activation and is regarded as a therapeutic target. Resveratrol inhibits ATC cell growth, while its impact on BRAF-MAPK signaling remains unknown. This study aims to address this issue by elucidating the statuses of BRAF-MAPK and STAT3 signaling activities in resveratrol-treated THJ-11T, THJ-16T, and THJ-21T ATC cells and Nthyori 3-1 thyroid epithelial cells. RT-PCR and Sanger sequencing revealed MKRN1-BRAF fusion mutation in THJ-16T, BRAF V600E point mutation in THJ-21T, and wild-type BRAF genes in THJ-11T and Nthyori 3-1 cells. Western blotting and immunocytochemical staining showed elevated pBRAF, pMEK, and pERK levels in THJ-16T and THJ-21T, but not in THJ-11T or Nthyori 3-1 cells. Calcein/PI, EdU, and TUNEL assays showed that compared with docetaxel and doxorubicin and MAPK-targeting dabrafenib and trametinib, resveratrol exerted more powerful inhibitory effects on mutant BRAF-harboring THJ-16T and THJ-21T cells, accompanied by reduced levels of MAPK pathway-associated proteins and pSTAT3. Trametinib- and dabrafenib-enhanced STAT3 activation was efficiently suppressed by resveratrol. In conclusion, resveratrol acts as dual BRAF-MAPK and STAT3 signaling inhibitor and a promising agent against ATCs with BRAF mutation.

Frequently expressed glypican-3 as a promising novel therapeutic target for osteosarcomas.

Osteosarcoma (OS) is the most common bone malignancy without a reliable therapeutic target. Glypican-3 (GPC3) mutation and upregulation have been detected in multidrug resistant OS, and anti-GPC3 immunotherapy can effectively suppress the growth of organoids. Further profiling of GPC3 mutations and expression patterns in OS is of clinical significance. To address these issues, fresh OS specimens were collected from 24 patients for cancer-targeted next-generation sequencing (NGS) and three-dimensional patient-derived organoid (PDO) culture. A tumor microarray was prepared using 37 archived OS specimens. Immunohistochemical (IHC) staining was performed on OS specimens and microarrays to profile GPC3 and CD133 expression as well as intratumoral distribution patterns. RT-PCR was conducted to semiquantify GPC3 and CD133 expression levels in the OS tissues. Anti-GPC3 immunotherapy was performed on OS organoids with or without GPC3 expression and its efficacy was analyzed using multiple experimental approaches. No OS cases with GPC3 mutations were found, except for the positive control (OS-08). IHC staining revealed GPC3 expression in 73.77% (45/61) of OSs in weak (+; 29/45), moderate (++; 8/45), and strong (+++; 8/45) immunolabeling densities. The intratumoral distribution of GPC3-positive cells was variable in the focal (+; 10%-30%; 8/45), partial (++; 31%-70%; 22/45), and the most positive patterns (+++; >71%; 15/45), which coincided with CD133 immunolabeling (P = 9.89 × 10-10 ). The anti-GPC3 antibody efficiently inhibits Wnt/ß-catenin signaling and induces apoptosis in GPC3-positive PDOs and PDXs, as opposed to GPC3-negative PDOs and PDXs. The high frequency of GPC3 and CD133 co-expression and the effectiveness of anti-wild-type GPC3-Ab therapy in GPC3-positive OS models suggest that GPC3 is a novel prognostic parameter and a promising therapeutic target for osteosarcoma.

Higher efficacy of resveratrol against advanced breast cancer organoids: A comparison with that of clinically relevant drugs.

The lack of reliable drugs is a therapeutic challenge of advanced breast cancers (ABCs). Resveratrol (Res) exerts inhibitory effects on breast cancer cell lines and animal models, while its efficacy against individual breast cancer cases remains unknown. This study aims to use ABC-derived organoids (ABCOs) as the ex vivo therapeutic platform to clarify the effectiveness of resveratrol against different ABC subtypes. Immunohistochemical staining confirmed that the ABCOs maintained their original tumors' ER, PR, HER2, and Ki67 expression patterns. ABCO proliferation and viability tests showed >50% cell death rates in 79.2% (19/24) of Res-treated, 28.6% (2/7) fulvestrant-treated, 66.7% (4/6) paclitaxel-treated, and 66.7% (6/9) gemcitabine-treated ABCOs. pSTAT3 nuclear translocation was more frequent in Res-sensitive (17/19; 89.47%) than that (1/5; 20%) of Res-insensitive ABCOs, which were suppressed upon Res treatment. Statistical analysis revealed a close correlation of STAT3 activation with the efficacy of Res, but not related to tumor receptor expression patterns (ER, PR, HER2) and pathological classification. We demonstrate for the first time the higher efficacy and broader spectrum of Res against different subtypes of ABCOs in comparison with that of conventional antibreast cancer drugs, providing an alternative approach for better management of ABCs.

Increased Reactive Oxygen Species and Distinct Oxidative Damage in Resveratrol-suppressed Glioblastoma Cells.

Background and Aim: Glioblastoma multiforme (GBM) is a highly aggressive brain malignancy that lacks reliable treatments. Resveratrol possesses anti-cancer effects, but its activity against glioblastoma cells is variable for unknown reasons. One mechanism through which anti-cancer drugs exert their effects is oxidative damage caused by increased reactive oxygen species (ROS) production. Thus, the present study examined the relationship between oxidative stress and sensitivity to resveratrol in glioblastoma cells. Methods: Two GBM cell lines (U251 and LN428) were exposed to 100 µM resveratrol for 48 h, and proliferation and apoptosis were assessed. ROS generation was evaluated using 2'-7'-dichlorodihydrofluorescein diacetate-based flow cytometry and fluorescent microscopy. Immunocytochemical staining and western blotting were conducted at regular intervals to profile the expression patterns of superoxide dismutase-2 (SOD2), catalase, caspase-9, caspase-3, and sulfotransferases (SULTs) in untreated and resveratrol-treated GBM cells. Results: Resveratrol-treated U251 cells, but not resveratrol-treated LN428 cells, exhibited remarkable growth arrest and extensive apoptosis accompanied by elevated intracellular ROS levels and attenuated SOD2 and catalase expression. Mitochondrial impairment and more distinct increases in the expression of activated caspase-9 and caspase-3 were detected in U251 cells following resveratrol treatment. The levels of resveratrol metabolic enzymes (SULT1A1 and SULT1C2) were lower in U251 cells than in LN428 cells. Conclusions: Resveratrol increased ROS generation and induced oxidation-related cellular lesions in U251 cells by activating an ROS-related mitochondrial signal pathway. The levels of SULTs and ROS may indicate the therapeutic outcomes of resveratrol treatment in GBM.

STAT3 signaling statuses determine the fate of resveratrol-treated anaplastic thyroid cancer cells.

BACKGROUNDS: Anaplastic thyroid cancer/ATC is highly lethal malignancy without reliable chemotherapeutic drug. Resveratrol possesses anti-ATC activities but encounters resistance in some cases due to certain unknown reason(s). OBJECTIVE: Because signal transducer and activator of transcription/STAT3 signaling is critical for ATC cell survival and the main molecular target of resveratrol, its roles in determining the fates of resveratrol-treated ATC cells were investigated here. METHODS: Human THJ-11T, THJ-16 and THJ-21T ATC cell lines were treated by 100 µM resveratrol and their growth, statuses of STAT3 signaling and STAT3-related gene expression were examined. The relevance of STAT3 activation with resveratrol resistance was elucidated using STAT selective inhibitor AG490. Leukemia inhibitory factor/LIF expression and phosphorylated-STAT3/p-STAT3 nuclear translocation in ATC tissues were immunohistochemically analyzed. RESULTS: Resveratrol inhibited proliferation, p-STAT3 nuclear translocation as well as LIF and STAT3 expression of THJ-16T and THJ-21T but not THJ-21T cells which showed LIF upregulation and more frequent p-STAT3 nuclear translocation. AG490 significantly prevent p-STAT3 nuclear translocation, and reversed the resveratrol tolerance of THJ-11T cells. Immonohistochemical staining revealed 14.3% (4/28) of LIF and 3.6% (1/28) of p-STAT3 detection in noncancerous ATC-surrounding tissues, which increased to 89.5% (17/19) and 52.6% (10/19) respectively among ATC specimens. The correlative analysis indicated the relevance of LIF expression and STAT3 activation (r= 0.825; P< 0.01). CONCLUSIONS: The status of STAT3 activation and LIF expression are closely correlated with the therapeutic effect of resveratrol on ATCs. Frequent LIF upregulation and STAT3 activation are the unfavorable factors of ATCs and the potential targets of anti-ATC therapy.

Resveratrol Reverses Retinoic Acid Resistance of Anaplastic Thyroid Cancer Cells via Demethylating CRABP2 Gene.

Background: Cellular retinoic acid binding protein 2 (CRABP2) mediates retinoic acid/RA anti-cancer pathways. Resveratrol effectively reverses RA tolerance and upregulates CRABP2 expression of anaplastic thyroid cancer cell line THJ-11T. As DNA methylation is responsible for CRABP2 silencing, the CRABP2 methylation status of THJ-11T cells and the demethylating effect of resveratrol on this gene are elucidated. Materials and methods: The statuses of CRABP2 expression and methylation and the levels of DNA methyltransferases (DNMTs) DNMT1, DNMT3A, and DNMT3B of THJ-11T cells were examined before and after resveratrol treatment via multiple experimental methods. The human medulloblastoma UW228-2 cell line was cited as the control of CRABP2 methylation and gemcitabine as the demethylator control. Results: RT-PCR, immunocytochemical staining and Western blotting showed that resveratrol significantly increased the CRABP2 expression and RA sensitivity of THJ-11T and UW228-2 cells. Bisulfite sequencing showed five CpG methylation sites at the CRABP2 promoter region of both cell lines, which were partially (3/5) demethylated by resveratrol and totally (5/5) by gemcitabine. DNMT1, DNMT3A, and DNMT3B were reduced in UW228-2 cells and DNMT1 and DNMT3A were reduced in THJ-11T cells after resveratrol treatment in a time-related fashion. Conclusion: Resveratrol is able to erase CRABP2 methylation and can thereby increase the RA sensitivity of THJ-11T and UW228-2 cells. This study demonstrates the additional value of the natural polyphenolic compound resveratrol as a demethylator in cancer treatments.

Efficacy and safety of intraperitoneally administered resveratrol against rat orthotopic ovarian cancers.

BACKGROUND: Resveratrol (Res) inhibits ovarian cancer (OC) cell growth but its in vivo anti-OC effects are unclear due to the low bioavailability of systemically administered Res. Intraperitoneal administration may overcome this therapeutic dilemma because it makes Res directly affect the abdominal tumors. Ethanol and DMSO are common Res solvents, while their reliability and safety for long-term in vivo treatment remain unknown. METHODS: A rat orthotopic OC model was established using the rat NUTU-19 OC cell line. Res dissolved in 10% ethanol or 0.2% DMSO was injected intraperitoneally (20 mg/kg/day) into tumor-free and tumor-bearing rats for 2 weeks. The tumors were collected for gross, morphological and molecular examinations, and blood and ascitic samples were obtained for a CA125 ELISA. Res concentration in ovarian tissues was determined by high performance liquid chromatography (HPLC). RESULTS: The average tumor weight (0.187±0.065 g) of the Res-in-DMSO group was lower than that of untreated (0.426±0.091 g; P<0.01) and Res-in-ethanol (0.238±0.073 g; P<0.05) group. The average bloody ascitic volumes collected from untreated, Res-in-ethanol, and Res-in-DMSO groups were 5.65±0.27, 2.75±0.14, and 2.09±0.11 ml, respectively. Abundant TUNEL-positive cells, ARHI and PIAS3 upregulation, CA125 reduction, and decreased STAT3 nuclear translocation were found in the Res-in-ethanol and, especially, the Res-in-DMSO group. Widespread plaques of Res deposits were found on the abdominal serosa of the Res-in-ethanol group, but not in the Res-in-DMSO group. HPLC revealed a higher Res concentration in Res-in-DMSO-treated tumor tissues than in those treated by Res-in-ethanol (P<0.01). Fertility was maintained after long-term Res treatment. CONCLUSION: Intraperitoneal administration of Res effectively inhibited rat orthotopic ovarian cancer growth without affecting normal tissues. The Res-in-DMSO group had the highest drug bioavailability and therefore stronger tumor-suppressive effects on ovarian cancer tissues.

Differential Exosomic Proteomic Patterns and Their Influence in Resveratrol Sensitivities of Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the commonest primary brain malignancy with extremely poor prognosis. Resveratrol posseses anti-cancer effects, while GBM cells respond differently to it due to certain unknown reason(s). Because the tumor-derived exosomes are supposed to influence chemosensitivity, the exosomic proteins released from resveratrol-sensitive U251 and resveratrol-resistant glioblastoma LN428 cells are profiled before (N/Exo) and after drug treatment (Res/Exo) by label-free liquid chromatography-mass spectrometry (LC-MS). The therapeutic implications of the proteomic findings are estimated by gene ontology enrichment analysis (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG)-based bioinformatic analyses and further elucidated by exosome co-incubating. The results reveal that U251/N/Exo but not U251/Res/Exo enhances resveratrol sensitivity of resveratrol-resistant LN428 cells. The resveratrol sensitive properties of U251 cells are not altered by either LN428/N/Exo or LN428/Res/Exo. U251/N/Exo contains higher levels of chromatin silencing and epidermis development proteins, while U251/Res/Exo has more oxygen transport and G protein-coupled receptor. Both of LN428/N/Exo and LN428/Res/Exo are rich in the proteins related with nucleosome assembly, microtubule-based process and chromatin silencing. In conclusion, U251/N/Exo sensitizes LN428 cells to resveratrol via delivering drug sensitizing signals, suggesting the presence of additional factor(s) that may determine the resveratrol sensitivities of glioblastoma cells.

Postoperative resveratrol administration improves prognosis of rat orthotopic glioblastomas.

BACKGROUND: Although our previous study revealed lumbar punctured resveratrol could remarkably prolong the survival of rats bearing orthotopic glioblastomas, it also suggested the administration did not completely suppress rapid tumour growth. These evidences led us to consider that the prognosis of tumour-bearing rats may be further improved if this treatment is used in combination with neurosurgery. Therefore, we investigated the effectiveness of the combined treatment on rat orthotopic glioblastomas. METHODS: Rat RG2 glioblastoma cells were inoculated into the brains of 36 rats. The rats were subjected to partial tumour removal after they showed symptoms of intracranial hypertension. There were 28 rats that survived the surgery, and these animals were randomly and equally divided into the control group without postoperative treatment and the LP group treated with 100 µl of 300 µM resveratrol via the LP route. Resveratrol was administered 24 h after tumour resection in 3-day intervals, and the animals received 7 treatments. The intracranial tumour sizes, average life span, cell apoptosis and STAT3 signalling were evaluated by multiple experimental approaches in the tumour tissues harvested from both groups. RESULTS: The results showed that 5 of the 14 (35.7%) rats in the LP group remained alive over 60 days without any sign of recurrence. The remaining nine animals had a longer mean postoperative survival time (11.0 ± 2.9 days) than that of the (7.3 + 1.3 days; p < 0.05) control group. The resveratrol-treated tumour tissues showed less Ki67 labelling, widely distributed apoptotic regions, upregulated PIAS3 expression and reduced p-STAT3 nuclear translocation. CONCLUSIONS: This study demonstrates that postoperative resveratrol administration efficiently improves the prognosis of rat advanced orthotopic glioblastoma via inhibition of growth, induction of apoptosis and inactivation of STAT3 signalling. Therefore, this therapeutic approach could be of potential practical value in the management of glioblastomas.

Correlation of Reactive Oxygen Species Levels with Resveratrol Sensitivities of Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid carcinoma (ATC) is the most lethal thyroid malignancy without a reliable therapeutic agent. Resveratrol possesses cancer-suppressive effects, while its effect(s) on ATC cells remains unknown. Because oxidative damage caused by increased reactive oxygen species (ROS) is one of the therapeutic effects of anticancer drugs and oxidative stress-caused mitochondria swelling is observed in resveratrol-treated cancer cells, the oxidative statuses and their relevance with resveratrol sensitivities are elucidated using THJ-16T and THJ-11T ATC cells established from two human anaplastic thyroid carcinoma cases. The results revealed that resveratrol-treated THJ-16T rather than THJ-11T cells showed remarkable growth arrest and extensive apoptosis accompanied with the elevated ROS generation and the attenuated superoxide dismutase 2 (SOD2) and catalase (CAT) levels. Mitochondrial impairment and the enhanced caspase-9/caspase-3 activation are found only in resveratrol-sensitive THJ-16T cells. Treatment with the antioxidant N-acetylcysteine (NAC) partly attenuated resveratrol-induced ROS generation and apoptosis of THJ-16T cells. The levels of resveratrol metabolic enzymes (SULT1A1 and SULT1C2) in THJ-16T cells were lower than those in THJ-11T cells and therefore reversely related with resveratrol sensitivities of ATC cells. Our findings demonstrate the ability of resveratrol to increase ROS generation and oxidative-related cellular lesions in resveratrol-sensitive THJ-16T cells presumably through activating the ROS-mitochondrial signal pathway. The levels of SULTs and ROS may reflect the response manners of ATC cells to resveratrol.

Preventive Potential of Resveratrol in Carcinogen-Induced Rat Thyroid Tumorigenesis.

Thyroid cancer (TC) is the most common endocrine malignancy without reliable preventive agent. Resveratrol possesses in vitro anti-TC activities; while its effect(s) on thyroid tumorigenesis remains unknown. This study aims to address this issue using DEN/MNU/DHPN-induced rat carcinogenesis model. 50 male Sprague-Dawley rats were separated into four groups as Group-1 (5 rats); normally fed; Group-2 (15 rats); DEN/MNU/DHPN treatment only; Group-3 (15 rats) and -4 (15 rats); DEN/MNU/DHPN treatment; followed by resveratrol intragastric (IG) injection and intraperitoneal (IP) injection; respectively; in two-day intervals for 30 weeks. The results revealed that the average resveratrol concentration in thyroid tissues was 1.278 ± 0.419 nmol/g in IG group and 1.752 ± 0.398 nmol/g in IP group. The final body weights of Group-3 and Group-4 were lighter than that (p > 0.05) of Group-1; but heavier than Group-2 (p < 0.05). TC-related lesions (hyperplasia and adenomas) were found in 53.3% of Group-2; 33.3% Group-3 and 26.7% Group-4. Lower serum carcino-embryonic antigen (CEA) and thyroglobulin (Tg) levels; down-regulated expression of IL-6 and cyclooxygenase-2 (COX-2); reduction of NF-κB/p65 nuclear translocation; and elevated IkBα expression were found in the thyroid tissues of Group-3 and Group-4 in comparison with that of Group-2. These results demonstrate that IG and IP administered resveratrol efficiently reduces the frequency and severity of DEN/MNU/DHPN-caused TC-related lesions and would be of values in thyroid tumor prevention.

Resveratrol Suppresses the Growth and Enhances Retinoic Acid Sensitivity of Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid cancer (ATC) is a highly lethal undifferentiated malignancy without reliable therapies. Retinoic acid (RA) has been employed to promote redifferentiation of thyroid cancers by increasing their I131 uptake and radio-sensitivity, but its effect(s) on ATCs has not yet been ascertained. Likewise, resveratrol induces cancer redifferentiation but, also in this case, its effects on ATCs remain unknown. These issues have been addresses in the current study using three human ATC cell lines (THJ-11T, THJ-16T, and THJ-21T) through multiple experimental approaches. The results reveal that RA exerts a small inhibitory effect on these cell lines. In comparison with normally cultured cells, the total cell number in resveratrol-treated THJ-16T and THJ-21T cultures significantly decreased (p < 0.05), and this effect was accompanied by reduced Cyclin D1 immuno-labeling, increased apoptotic fractions, and distinct caspase-3 activation. Resveratrol failed to inhibit growth but enhanced RA sensitivity of THJ-11T cells, suppressed peroxisome proliferator-activated receptor-ß/δ (PPAR-ß/δ), and upregulated cellular retinoic acid-binding protein 2 (CRABP2) and retinoic acid receptor beta (RAR-ß) expression. Increased thyroglobulin (Tg) and E-cadherin levels and appearance of membranous E-cadherin were evidenced in resveratrol-treated THJ-11T cells. Our results demonstrate for the first time: (1) the therapeutic value of resveratrol by itself or in combination with RA in the management of ATCs, (2) the capacity of resveratrol to overcome RA resistance in ATC cells by reprogramming CRABP2/RAR- and fatty acid-binding protein 5 (FABP5)/PPAR-ß/δ-mediated RA signaling, and (3) the redifferentiating potential of resveratrol in ATC cells.

Inactivated Wnt signaling in resveratrol-treated epidermal squamous cancer cells and its biological implication.

Squamous cell carcinoma (SCC) is the most common epidermal malignancy, and Wnt/ß-catenin signaling is frequently activated in SCC. Resveratrol prevents rodent epidermal carcinogenesis, while its effect on human epidermal cancer remains unknown. To address this issue, the impact of resveratrol on the growth and Wnt signaling of skin SCC Colo16 cells were investigated at the cellular and molecular biology levels by flow cytometry, immunocytochemistry, reverse transcription-polymerase chain reaction, western blotting and ß-catenin-specific small interfering RNA (siRNA) transfection. Resveratrol (100 µM) suppressed cell growth and induced apoptosis in Colo16 cells. Wnt2 and its downstream genes were downregulated, which was accompanied by increased expression of the Wnt signaling inhibitor Axin2. Transfection with a ß-catenin-specific siRNA did not affect cell growth but enhanced the resveratrol susceptibility of Colo16 transfectants. The present results suggest the inhibitory effects of resveratrol on epidermal SCCs and inactivation of Wnt signaling as one of the resveratrol-caused molecular events in Colo16 cells. ß-catenin oriented siRNA is insufficient to induce cell crisis, implicating the presence of more critical cancer-associated element(s) as the target in Colo16 cells.

Correlative analyses of the expression levels of PIAS3, p-SHP2, SOCS1 and SOCS3 with STAT3 activation in human astrocytomas.

The importance of signal transducer and activator of transcription 3 (STAT3) signaling in the growth and survival of glioblastoma cells has been well documented, while the reasons leading to STAT3 activation remains to be elucidated. Suppressors of cytokine signaling (SOCS) 1 and SOCS3, SH2 domain­containing phosphatase (SHP2) and protein inhibitors of activated STAT3 (PIAS3) are known to inhibit STAT3 signal transduction, while their expression statuses in the four grades of astrocytomas and relevance with STAT3 activation remain to be described. The present study aimed to address these issues by tissue microarray­based immunohistochemical profiling the expression levels of phosphorylated (p)­STAT3, SOCS1, SOCS3, PIAS3 and p­SHP2. The results revealed that p­STAT3 nuclear translocation was rarely observed in non­cancerous brain tissues and its frequencies were increased in a tumor grade­associated manner (65.2, 77.1, 81.8 and 85.7% for grade I­IV, respectively). PIAS3, p­SHP2, SOCS1 and SOCS3 were expressed in higher levels (++ and +++) in 63.6, 90, 87.5 and 81.8% of tumor surrounding brain tissues, which reduced to 13.1, 47.8, 33.3 and 50% in grade I, 11.4, 65.7, 58.3 and 77.1% in grade II, 9.1, 63.6, 38.1 and 31.8% in grade III and 7.1, 66.7, 30.8 and 7.1% in grade IV astrocytomas. The above results revealed that although the expression levels of SOCS1, SOCS3 and, in particular, p­SHP2, tend to decrease in the four types of astrocytomas, PIAS3 downregulation is more negatively correlated with STAT3 activation in the stepwise progress of astrocytomas and would indicate an unfavorable outcome.

PIAS3, SHP2 and SOCS3 Expression patterns in Cervical Cancers: Relevance with activation and resveratrol-caused inactivation of STAT3 signaling.

OBJECTIVE: Resveratrol inhibits cervical cancer (CC) cells by blocking STAT3 signaling. However, the mechanism of resveratrol-induced STAT3 inactivation remains largely unknown. SHP2, PIAS3, and SOCS3 are STAT3 negative regulators; therefore, their statuses in cervical adenocarcinoma (HeLa) and squamous cell carcinoma (SiHa and C33A) cell lines without and with resveratrol treatment and their correlation with STAT3 activation in CC specimens were investigated. METHODS: MTT and TUNEL assays were used to check the resveratrol sensitivity of CC cells, and immunocytochemical staining, Western blotting, and RT-PCR were used to analyze SHP2, PIAS3, and SOCS3 expression and the intracellular distribution of STAT3. Tissue microarray based immunohistochemical staining was performed to investigate potential correlations between SHP2, PIAS3, and SOCS3 expression and STAT3 activation. RESULTS: PIAS3 and SOCS3 were found to be weakly expressed in CC cells and upregulated by resveratrol; this was accompanied by inhibition of STAT3 signaling. The SHP2 level remained unchanged in all three cell lines after resveratrol treatment. STAT3 nuclear translocation was more frequent in adenocarcinomas and squamous cell carcinomas than that of their noncancerous counterparts. The SOCS3 level and detection rate were higher in noncancerous squamous cells (but not in glandular epithelia) compared with their cancerous counterparts. The phospho-SHP2 detection rate was similar in noncancerous and tumor tissues of squamous and glandular origins; however, PIAS3 levels were distinct. CONCLUSIONS: Of the three STAT3 negative regulators, PIAS3 correlated most negatively with STAT3 nuclear translocation and may inhibit STAT3 signaling in both histological CC subtypes. PIAS3 responsiveness may reflect greater resveratrol sensitivity and improved therapeutic outcome in CCs.

Inhibition of STAT3 signaling as critical molecular event in resveratrol-suppressed ovarian cancer cells.

BACKGROUND: Resveratrol exerts inhibitory effects on ovarian cancer cells, while its underlying mechanism and critical molecular target(s) have been lesser known. Activations of Wnt, Notch and STAT3 signaling are frequent in ovarian cancers/OCs and supposed to be important for OC formation and progression, while the impacts of resveratrol on these signaling pathways in OC cells remain obscure. METHODS: In this study, two human ovarian cancer cell lines, OVCAR-3 and CAOV-3, were treated by 120 µM resveratrol and their responses to the treatment and the statuses of Wnt, Notch and STAT3 signaling in them were analyzed by multiple experimental approaches. Selective inhibitors of Wnt, Notch or STAT3 signaling were employed to treat OVCAR-3 and CAOV-3 cells to elucidate the significance of individual signaling pathways for ovarian cancers. RESULTS: The results demonstrated distinct inhibitory effects of resveratrol on human ovarian cancer cells in terms of remarkable G1 phase accumulation, increased apoptosis fraction and concurrent suppression of Wnt, Notch and STAT3 signaling as well as their downstream cancer-related gene expression. Treatments with Wnt, Notch or STAT3 selective inhibitor revealed that only AG490, a JAK-specific inhibitor, inhibits OVCAR-3 and CAOV-3 cells in the extent as similar as that of resveratrol. CONCLUSION: Our results suggest the significance of STAT3 activation in the maintenance and survival of ovarian cancer cells. The activated STAT3 signaling is the critical molecular target of resveratrol. Resveratrol would be a promising candidate in the management of ovarian cancers, especially the ones with resistance to conventional therapeutic agents.

CRABP-II- and FABP5-independent responsiveness of human glioblastoma cells to all-trans retinoic acid.

Glioblastomas respond differently to all-trans retinoic acid (RA) for unknown reasons. Because CRABP-II and FABP5 mediate RA intracellular signaling respectively and lead to distinct biological consequences, their expression patterns in different grades of astrocytomas and the glioblastoma cells lines LN18, LN428 and U251 were examined to identify potential correlations with RA sensitivities. The response of glioblastoma cells to RA, decitabine or the FABP5 competitive inhibitor, BMS309403, was analyzed. CRABP-II and FABP5 were expressed to varying degrees by the 84-astrocytoma cases examined. Treatment of LN428, U251 and LN18 cells with RA failed to suppress their growth; however, U251 proliferation was inhibited by decitabine. The combination of decitabine and RA suppressed the growth of all three cell lines and induced significant apoptosis of LN428 and U251 cells. Both CRABP-II and FABP5 were transcribed in the three cell lines but FABP5 proteins were undetectable in U251 cells. The ratio of CRABP-II to FABP5 was not altered after RA, decitabine or RA and decitabine treatment and the resistance of cells to RA was not reversed by BMS309403 treatment. In conclusion, CRABP-II and FABP5 expression patterns are neither related to the tumor grades nor correlated with RA sensitivity. Additional molecular factors may be present that determines the sensitivity of glioblastoma cells to RA. Dicitabine may improve the sensitivity of glioblastoma cells to RA, however, its underlying mechanism and its in vivo feasibility need to be investigated.

Diffusion Efficiency and Bioavailability of Resveratrol Administered to Rat Brain by Different Routes: Therapeutic Implications.

Resveratrol possesses anti-tumor activities against central nervous system (CNS) tumors in vitro but has not yet been used clinically due to its low bioavailability, particularly in the CNS. This study thus aimed to elucidate brain bioavailability of trans-resveratrol by monitoring brain concentrations and dwell times following administration of resveratrol through intragastric, intraperitoneal, external carotid artery/ECA and intrathecal routes. In parallel, we evaluated the biological responses of rat RG2 glioblastoma cells as well as RG2-formed rat intracranial glioblastomas treated with resveratrol via intrathecal administration. The results revealed that resveratrol was detected in rat brains except when administered systemically. Intrathecal administration of reseveratrol led to abundant apoptotic foci and increased staining of the autophagy proteins, LC-3 and Beclin-1 and shrinkage of the intracranial tumors. In conclusion, the BBB penetrability of resveratrol is remarkably increased by intracthecal administration. Regular short-term resveratrol treatments suppress growth and enhance autophagic and apoptotic activities of rat RG2 glioblastoma cells in vitro and in vivo. Therefore, intrathecal administration of resveratrol could be an optimal intervention approach in the adjuvant management of brain malignancies.