A nomogram prediction model for lymph node metastasis in endometrial cancer patients.

BACKGROUND: This study aimed to explore the risk factors for lymph node metastasis (LNM) in patients with endometrial cancer (EC) and develop a clinically useful nomogram based on clinicopathological parameters to predict it. METHODS: Clinical information of patients who underwent staging surgery for EC was abstracted from Qilu Hospital of Shandong University from January 1st, 2005 to June 31st, 2019. Parameters including patient-related, tumor-related, and preoperative hematologic examination-related were analyzed by univariate and multivariate logistic regression to determine the correlation with LNM. A nomogram based on the multivariate results was constructed and underwent internal and external validation to predict the probability of LNM. RESULTS: The overall data from the 1517 patients who met the inclusion criteria were analyzed. 105(6.29%) patients had LNM. According the univariate analysis and multivariate logistic regression analysis, LVSI is the most predictive factor for LNM, patients with positive LVSI had 13.156-fold increased risk for LNM (95%CI:6.834-25.324; P < 0.001). The nomogram was constructed and incorporated valuable parameters including histological type, histological grade, depth of myometrial invasion, LVSI, cervical involvement, parametrial involvement, and HGB levels from training set. The nomogram was cross-validated internally by the 1000 bootstrap sample and showed good discrimination accuracy. The c-index for internal and external validation of the nomogram are 0.916(95%CI:0.849-0.982) and 0.873(95%CI:0.776-0.970), respectively. CONCLUSIONS: We developed and validated a 7-variable nomogram with a high concordance probability to predict the risk of LNM in patients with EC.

Iliac ecchymosis, a valuable sign for hollow viscus injuries in blunt pelvic trauma patients.

PURPOSE: Pelvic fractures are characterized by high energy injuries and often accompanied with abdominal and pelvic organ injury. CT has been applied for several decades to evaluate blunt pelvic trauma patients. However, it has a certain rate of inaccurate diagnosis of abdominal hollow viscus injury (HVI), especially in the early stage after injury. The delayed diagnosis of HVI could result in a high morbidity and mortality. The bowel injury prediction score (BIPS) applied 3 clinical variables to determine whether an early surgical intervention for blunt HVI was necessary. We recently found another clinical variable (iliac ecchymosis, IE) which appeared at the early stage of injury, could be predicted for HVI. The main objective of this study was to explore the novel combination of IE and BIPS to enhance the early diagnosis rate of HVI, and thus reduce complications and mortalities. METHODS: We conducted a retrospective analysis from January 2008 to December 2018 and recorded blunt pelvic trauma patients in our hospital. The inclusion criteria were patients who were verified with pelvic fractures using abdomen and pelvis CT scan in the emergency department before any surgical intervention. The exclusion criteria were abdominal CT insufficiency before operation, abdominal surgery before CT scan, and CT mesenteric injury grade being 5. The MBIPS was defined as BIPS plus IE, which was calculated according to 4 variables: white blood cell counts of 17.0 or greater, abdominal tenderness, CT scan grade for mesenteric injury of 4 or higher, and the location of IE. Each clinical variable counted 1 score, totally 4 scores. The location and severity of IE was also noted. RESULTS: In total, 635 cases were hospitalized and 62 patients were enrolled in this study. Of these included patients, 77.4% (40 males and 8 females) were operated by exploratory laparotomy and 22.6% (8 males and 6 females) were treated conservatively. In the 48 patients underwent surgical intervention, 46 were confirmed with HVI (45 with IE and 1 without IE). In 46 patients confirmed without HVI, only 3 patients had IE and the rest had no IE. The sensitivity and specificity of IE in predicting HVI was calculated as 97.8% (45/46) and 81.3% (13/16), respectively. The median MBIPS score for surgery group was 2, while 0 for the conservative treatment group. The incidence of HVI in patients with MBIPS score ≥ 2 was significantly higher than that in patients with MBIPS score less than ≤ 2 (OR = 17.3, p < 0.001). CONCLUSION: IE can be recognized as an indirect sign of HVI because of the high sensitivity and specificity, which is a valuable sign for HVI in blunt pelvic trauma patients. MBIPS can be used to predict HVI in blunt pelvic trauma patients. When the MBIPS score is ≥ 2, HVI is strongly suggested.

CD271 antibody-functionalized HGNs for targeted photothermal therapy of osteosarcoma stem cells.

Cancer stem cells (CSCs) are considered to maintain the vitality of tumor cell populations through self-renewal and infinite proliferation, but their accessibility is still under investigation. In addition, CSCs are more resistant to chemotherapy and radiotherapy compared with common tumor cells. This study aimed to develop a kind of novel and feasible nanomaterial for targeted photothermal ablation of osteosarcoma stem cells, which could be a promising anticancer strategy. The osteosarcoma stem cells were extracted by serum-free culture and we further verified the stem cell properties. We evaluated the expression of CD271 by flow cytometry. PEGylated multifunctional hollow gold nanospheres (HGNs) were prepared based on CD271 monoclonal antibody. Bifunctional SH-PEG-COOH was used to facilitate the covalent linkage between HGNs and antibody. The efficient uptake and distribution of the functionalized HGNs were investigated using ICP-MS and TEM. Morphological studies and quantitative apoptosis evaluation were performed to detect the effect of photothermal therapy (PTT). Afterwards, we explored the possible mechanism by which PTT induced targeted killing of cancer stem cells. Osteosarcoma cells isolated from serum-free culture were detected to show stem cell properties. CD271 was found to be a potential novel surface marker for osteosarcoma stem cells. By conjugating with CD271 monoclonal antibody, these biomimetic nanoparticles can be targeted and absorbed by osteosarcoma stem cells. HGNs-PEG-CD271 achieved excellent cell viability inhibition compared with non-targeted PEGylated HGNs upon near-infrared (NIR) laser irradiation. The mechanism of targeted killing may be related to the apoptosis pathway and DNA double-strand injuries. CD271 was considered to be a surface biomarker for osteosarcoma stem cells. Functionalized HGNs based on CD271 antibody exhibited excellent potential for targeted PTT, which may be a promising strategy for osteosarcoma treatment.

Assay of Peripheral Regulatory Vδ1 T Cells in Ankylosing Spondylitis and its Significance.

BACKGROUND Ankylosing spondylitis (AS) involves inflammation at the sacroiliac joint and spine attachment site. This study aimed to observe the ratio and function of peripheral regulatory Vδ1 T cells in AS patients to investigate their roles in AS pathogenesis. MATERIAL AND METHODS Peripheral blood mononuclear cells (PBMC) were separated by density-gradient centrifugation from AS patients and healthy controls. Flow cytometry was used to determine the ratio between Vδ1 and CD4 T cells of PBMC in AS patients and controls. Flow cytometry sorting (FCS) was used to obtain Vδ1 and naïve CD4 T cells with purity higher than 90%. CFSE staining method was used to detect the effect of Vδ1 T cells on proliferation of naïve CD4 T cells. The effect of Vδ1 T cells on secretion of IFN-γ from naïve CD4 T cells and the ability to secrete IL-10 from Vδ1 T cells were determined by flow cytometry. RESULTS AS patients had significantly lower Vδ1 T cell ratio in PBMC compared to controls (p<0.05), but their CD4 T cell ratio was significantly elevated (p<0.05). Functional assay showed suppression of naïve CD4 T cell proliferation and IFN-γ secretion by peripheral Vδ1 T cells in AS patients (p<0.01). AS patients also had lower IL-10 secreting level from peripheral derived Vδ1 T cells (p<0.01). CONCLUSIONS The immune suppression of peripheral Vδ1 T cell in AS patient increases the ratio of peripheral CD4 T cells and IFN-γ level, leading to AS pathogenesis. This immune suppression is mainly due to suppressed IL-10 secretion.

Downregulation of DNA-PKcs suppresses P-gp expression via inhibition of the Akt/NF-κB pathway in CD133-positive osteosarcoma MG-63 cells.

The development of chemoresistance is closely linked to the plateau of the survival rate in osteosarcoma (OS) patients. CD133-positive (CD133+) OS cells are known as cancer stem cells (CSCs) in OS and exhibit the characteristic of chemoresistance. In this study, CD133+ and CD133­negative (CD133­) MG­63 cells were isolated by magnetic activated cell sorting (MACS). We verified that CD133+ MG­63 cells were more resistant to cisplatin (CDDP) than CD133­ MG­63 cells. DNA­dependent protein kinase catalytic subunit (DNA­PKcs) and P­glycoprotein (P­gp) were expressed at higher levels in the CD133+ MG­63 cells compared with those levels in the CD133­ MG­63 cells, whereas downregulation of DNA­PKcs by small interfering RNA (siRNA) decreased chemoresistance to CDDP and P­gp expression at the mRNA and protein levels in these cells. This indicated that DNA­PKcs was correlated with P­gp expression in the CD133+ MG­63 cells. The Akt/NF­κB pathway was hyperactivated in the CD133+ MG­63 cells, whereas inhibition of the Akt/NF­κB pathway downregulated P­gp expression. In addition, downregulation of DNA­PKcs suppressed the activity of the Akt/NF­κB pathway. These results revealed that downregulation of DNA­PKcs could decrease P­gp expression via suppression of the Akt/NF­κB pathway in CD133+ MG­63 cells. Therefore, inhibition of DNA­PKcs decreases P­gp expression and sensitizes OS CSCs to chemotherapeutic agents in vitro, which needs to be further validated in vivo.

Diallyl trisulfide induces osteosarcoma cell apoptosis through reactive oxygen species-mediated downregulation of the PI3K/Akt pathway.

Diallyl trisulfide (DATS) is a natural organosulfur compound isolated from garlic, and has been reported to possess anticancer activities. However, the cancer growth inhibitory effects and molecular mechanisms in human osteosarcoma cells have not been well studied. The present study demonstrated that DATS significantly reduced cell viability in a dose- and time-dependent manner in MG63 and MNNG/HOS cells. DATS-induced G0/G1 phase arrest was found to correlate with a decrease in cyclin D1 in concomitance with an increase in p21 and p27. DATS induced a marked increase in reactive oxygen species (ROS) levels and collapse of mitochondrial membrane potential (Δψm) in the osteosarcoma cells. DATS induced apoptosis in the MG63 and MNNG/HOS cells via inhibition of the PI3K/Akt signaling pathway and through the mitochondrial apoptotic pathway. The efficiency of DATS basically approached the efficacy of LY294002, a specific PI3K inhibitor. However, N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the DATS-induced ROS increase, inhibition of the PI3K/Akt pathway and cell apoptosis. Overall, DATS has the potential to be developed as a new anticancer drug. The mechanisms of action involve the ROS-mediated downregulation of the PI3K/Akt pathway.

Targeting DNA-PKcs increased anticancer drug sensitivity by suppressing DNA damage repair in osteosarcoma cell line MG63.

Many chemotherapy drugs exert anticancer effects through causing DNA damage, such as DNA topoisomerase inhibitor and platinum-containing drugs. DNA damage repair is an important mechanism of drug resistance which is responsible for metastasis and recurrence after chemotherapy. DNA-dependent protein kinase (DNA-PK) plays an important role in non-homology end joining (NHEJ) pathway. In this study, we aimed to determine whether DNA-PK catalytic subunit (DNA-PKcs) is expressed in osteosarcoma MG63 cell line and involved in drug resistance induced by DNA repair. We found that DNA-PKcs was expressed in osteosarcoma cell line MG63. The pDNA-PKcs(T2609) was more expressed in cells treated with cisplatin (DDP) and etoposide (VP16). Down-regulation of DNA-PKcs produced higher sensitivity of MG63 cells to DDP or VP16 through increasing apoptosis and causing cell cycle arrest in the G1 phase. Our study supported that DNA-PKcs was involved in drug-induced DNA damage repair and related to chemosensitivity of osteosarcoma MG63 cells.

Anti-TROP2 conjugated hollow gold nanospheres as a novel nanostructure for targeted photothermal destruction of cervical cancer cells.

Photothermal ablation (PTA) is a promising avenue in the area of cancer therapeutics that destroys tumor cells through conversion of near-infrared (NIR) laser light to heat. Hollow gold nanospheres (HGNs) are one of the few materials that are capable of converting light to heat and have been previously used for photothermal ablation studies. Selective delivery of functional nanoparticles to the tumor site is considered as an effective therapeutic approach. In this paper, we demonstrated the anti-cancer potential of HGNs. HGNs were conjugated with monoclonal antibody (anti-TROP2) in order to target cervical cancer cells (HeLa) that contain abundant trophoblast cell surface antigen 2 (TROP2) on the cell surface. The efficient uptake and intracellular location of these functionalized HGNs were studied through application of inductively coupled plasma atomic emission spectroscopy (ICP-AES) and transmission electron microscopy (TEM). Cytotoxicity induced by PTA was measured using CCK-8 assay. HeLa cells incubated with naked HGNs (0.3-3 nmol L(-1)) within 48 h did not show obvious cytotoxicity. Under laser irradiation at suitable power, anti-TROP2 conjugated HGNs achieved significant tumor cell growth inhibition in comparison to the effects of non-specific PEGylated HGNs (P < 0.05). γH2AX assay results revealed higher occurrences of DNA-DSBs with anti-TROP2 conjugated HGNs plus laser radiation as compared to treatment with laser alone. Flow cytometry analysis showed that the amount of cell apoptosis was increased after laser irradiation with anti-TROP2 conjugated HGNs (P < 0.05). Anti-TROP2 conjugated HGNs resulted in down-regulation of Bcl-2 expression and up-regulation of Bax expression. Our study results confirmed that anti-TROP2 conjugated HGNs can selectively destroy cervical cancer cells through inducing its apoptosis and DNA damages. We propose that HGNs have the potentials to mediate targeted cancer treatment.

CD271+ osteosarcoma cells display stem-like properties.

Cancer stem cell (CSC) theory has been proposed and verified in many cancers. The existence of osteosarcoma CSCs has been confirmed for many years and multiple surface markers have been employed to identify them. In this study, we identified CD271(+) subpopulation of osteosarcoma displaying stem-like properties. CD271, known as the neural crest nerve growth factor receptor, is the marker of bone marrow mesenchymal stem cells (MSCs) and human melanoma-initiating cells. We discovered that CD271 was expressed differentially in diverse types of human osteosarcoma and stabilized cell lines. CD271(+) osteosarcoma cells displayed most of the properties of CSC, such as self-renewal, differentiation, drug resistance and tumorigenicity in vivo. Nanog, Oct3/4, STAT3, DNA-PKcs, Bcl-2 and ABCG2 were more expressed in CD271(+) cells compared with CD271- cells. Our study supported the osteosarcoma CSC hypothesis and, to a certain extent, revealed one of the possible mechanisms involved in maintaining CSCs properties.

Reversion effects of curcumin on multidrug resistance of MNNG/HOS human osteosarcoma cells in vitro and in vivo through regulation of P-glycoprotein.

BACKGROUND: P-glycoprotein (P-gp) encoded by ATP-binding cassette sub-family B member 1 (ABCB1) gene is a kind of ATP-dependent drug transporter, which plays important roles in multidrug resistance (MDR) of human cancers, such as osteosarcoma. Curcumin is a natural phenolic coloring compound originating from the rhizomes of Curcuma longa, which is proved to possess antitumor biological activities including reversion of MDR. However, the effect and molecular mechanisms of curcumin to osteosarcoma MDR remain unclear. METHODS: We established a human osteosarcoma drug-resistant cell line MNNG/HOS/MTX by pulse exposure to methotrexate (MTX) and verified that the new cell lines were cross-resistant to other anticancer agents. Then, according to the cytotoxicity assay, we reversed MDR of MNNG/HOS/MTX by 30 µmol/L curcumin, and detected the mechanisms of curcumin reversing MDR through Real-time PCR, Western blotting assay, and Rhodamine123 (Rh123) transport test. Finally, we evaluated the effect of curcumin reversing MDR in vivo by MNNG/HOS/MTX cells xenograft-nude mice model. RESULTS: MNNG/HOS/MTX was proved to be a human osteosarcoma MDR cell line. MTT tumor chemosensitivity test indicates that 30 µmol/L curcumin attenuates the half maximal inhibitory concentration (IC50) and resistance index (RI) to MTX, diamminedichloroplatinum (DDP), adriamycin (ADM), ifosfamide (IFO), and epirubicin (EPI) in MNNG/HOS/MTX cells (P < 0.05). Real-time PCR and Western blotting assays demonstrated that curcumin down-regulated P-gp expression of MNNG/HOS/MTX cells. Rh123 transport test showed that curcumin inhibited the transport function of P-gp in vitro. In vivo studies showed that curcumin displayed the features of sensitizing antitumor drugs and inhibiting the proliferation, invasion, and metastasis of osteosarcoma MDR cells. CONCLUSION: Down-regulation of P-gp and inhibition of the function of P-gp efflux pump may contribute to MDR reversion induced by curcumin in vitro and in vivo.

Overexpression of TROP2 predicts poor prognosis of patients with cervical cancer and promotes the proliferation and invasion of cervical cancer cells by regulating ERK signaling pathway.

Overwhelming evidence has demonstrated that the aberrant expression of the human trophoblast cell-surface antigen (TROP2) was associated with tumor aggressiveness and poor prognosis in a variety of human cancers, however the roles of TROP2 in cervical cancer have not been investigated. The purpose of our study was to elucidate the prognostic significance of TROP2 expression in patients with cervical cancer and determine its effect on tumor progression. Immunohistochemistry assay showed that 88.7% (94/106 cases) of cervical cancer specimens were positively stained with TROP2, and the overexpression of TROP2 was closely related with FIGO stage, histological grades, lymphatic metastasis, invasive interstitial depth and high expression of Ki-67. Patients with TROP2-positive staining exhibited a significantly decreased overall survival and progression free survival; it was also an independent predictor for prognosis according to multivariate analysis. Moreover, down-regulation of TROP2 mediated by siRNA in Siha and CaSki cells resulted in a strong inhibition of proliferation and invasion, TROP2 abrogation also elevated the apoptotic ratio and caused G1 arrest. Conversely, enforced expression of TROP2 in HeLa and C33A cells remarkably promoted cell growth, migration and invasion. In addition, the tumorigenic function of TROP2 was associated with the increased expressions of cyclin D1, cyclin E, CDK2 and CDK4 but reduced expression of p27 and E-cadherin via the activation of Erk1/2 signaling pathway. Furthermore, the inhibition of TROP2 expression in cervical cancer cell lines enhances sensitivity to cisplatin. The present study suggest that overexpression of TROP2 may play crucial roles in the development and pathogenesis of human cervical cancer, therefore, TROP2 may represent a prospective prognostic indicator and a potential therapeutic target of cervical cancer.