A preliminary study on the association between epithelial-mesenchymal transition and circulating tumor cells in renal cell carcinoma.

Background: Circulating tumor cells (CTCs) are considered useful prognostic factors for various cancers, and in 2014, our research group conducted a comparative experiment of CTC detection in patients with renal cell cancer (RCC). However, the reason for the low detection rate of CTCs in cancer patients using the CellSearch® system is still unknown, although it has been hypothesized to be attributed to the likelihood that CTCs undergoing epithelial-mesenchymal transition (EMT) do not express the CTC biomarkers cytokeratin (CK)8/18/19 or epithelial cell adhesion molecule (EpCAM). The overall aim of the current study was to investigate the expression levels of CK8/18/19 and EpCAM in relation to the EMT biomarkers vimentin and E-cadherin in patients with RCC. Methods: Patients with RCC who had undergone radical nephrectomy or partial resection between May 2014 and December 2014 were initially recruited. Results: Among 34 RCC patients, nine co-expressed EpCAM and CK8/18/19 in primary tumor tissues. The CellSearch® results showed that CK8/18/19 was expressed in 5 of 6 patients (5/6) and EpCAM was expressed in 6 patients (6/6). However, the isolation by size of tumor cells (ISET) technique showed these were co-expressed in only four of the 10. The expression of CK8/18/19, EpCAM, vimentin, and E-cadherin was distributed unequally in different enumeration groups of CTCs (all P>0.05), and the positive expression of CK8/18/19 was correlated with neutrophil number and tumor size (P<0.05). The positive expression of vimentin was correlated with the Karnofsky Performance Status (KPS) score and clinical stage of renal cancer patients (P<0.05). Conclusions: Our results indirectly proved the occurrence of EMT in the formation of CTCs by comparing and analyzing the expression of CK8/18/19 and EpCAM in renal cancer tissues and the detection results of CTCs.

Subclinical Lesions of the Primary Clinical Target Volume Margin in Esophageal Squamous Cell Carcinoma and Association With FDG PET/CT.

Background and Objectives: An accurate delineation of the primary clinical target volume (CTVp) in esophageal squamous cell carcinoma (ESCC) significantly affects the outcomes of radiotherapy. However, when basing the CTVp on the primary gross tumor volume, there are no consistent guidelines for the size of the margin. We compared preoperative 18F-fluorodeoxyglucose (FDG) PET/CT images and large slices of resected pathological ESCC specimens for evidence and prediction of subclinical lesions. We also investigated associations between the maximum standardized uptake value (SUVmax), metabolic tumor volumes (MTVs), and lesions to improve estimates of the CTVp. Methods:55 patients underwent FDG PET/CT before surgery, and the SUVmax and MTVs were determined. To ensure that the in situ distances between the primary and secondary tumors were preserved, the esophageal specimens collected during radical surgery were processed to minimize shrinkage, and subclinical lesions were characterized by pathological examination. A 2-dimensional logistic regression model was used to assess the associations between clinicopathological features and microscopic spread of the lesions. Results: Subclinical lesions in pathological specimens were characterized as direct invasion, multicentric occurrence lesions, intra-mural metastasis, vascular invasion, and perineural invasion in 56.4, 40.0, 30.9, 21.8, and 18.2% of patients, respectively. The mean distances of the subclinical lesions from the primary tumor were 0.79 ± 1.28 cm and 0.87 ± 1.00 cm in the cranial and caudal directions, respectively. Together the SUVmax and MTV values could predict the presence of subclinical lesions that were not detectable in PET/CT images. Conclusions: To cover 94.5% of ESCC subclinical lesions in the CTVp, a 3-cm margin along the cranial-caudal axis should be added to the primary gross tumor volume as defined by FDG-PET/CT, as well as a cutoff SUVmax value of 2.5. Although preoperative FDG PET/CT images may not reveal lesions directly, the SUVmax and MTV measurements together could predict their presence.

Hispidulin induces ER stress-mediated apoptosis in human hepatocellular carcinoma cells in vitro and in vivo by activating AMPK signaling pathway.

Hispidulin (4',5,7-trihydroxy-6-methoxyflavone) is a phenolic flavonoid isolated from the medicinal plant S. involucrata, which exhibits anti-neoplastic activity against several types of cancer. However, the mechanism underlying its anti-cancer activity against hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, we investigated whether and how hispidulin-induced apoptosis of human HCC cells in vitro and in vivo. We showed that hispidulin (10, 20 µmol/L) dose-dependently inhibited cell growth and promoted apoptosis through mitochondrial apoptosis pathway in human HCC SMMC7721 cells and Huh7 cells. More importantly, we revealed that its pro-apoptotic effects depended on endoplasmic reticulum stress (ERS) and unfolded protein response (UPR), as pretreatment with salubrinal, a selective ERS inhibitor, or shRNA targeting a UPR protein CHOP effectively abrogated hispidulin-induced cell apoptosis. Furthermore, we showed that hispidulin-induced apoptosis was mediated by activation of AMPK/mTOR signaling pathway as pretreatment with Compound C, an AMPK inhibitor, or AMPK-targeting siRNA reversed the pro-apoptotic effect of hispidulin. In HCC xenograft nude mice, administration of hispidulin (25, 50 mg/kg every day, ip, for 27 days) dose-dependently suppressed the tumor growth, accompanied by inducing ERS and apoptosis in tumor tissue. Taken together, our results demonstrate that hispidulin induces ERS-mediated apoptosis in HCC cells via activating the AMPK/mTOR pathway. This study provides new insights into the anti-tumor activity of hispidulin in HCC.

Using 18F-fluorodeoxyglucose positron emission tomography/computed tomography to estimate the length of gross tumor and involvement of lymph nodes in esophagogastric junction carcinoma.

OBJECTIVE: To determine the optimal approach for estimating the length of gross tumor and involvement of the lymph nodes with 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) in esophagogastric junction carcinoma (EGJC). The result was verified with pathologic examination. MATERIALS AND METHODS: Twenty patients with diagnosed and untreated EGJC were enrolled. The length of the gross tumor was measured using different approaches with PET/CT: Standardized uptake value (SUV) 1.5-5.5 in intervals of 1.0 and 10%-50% of maximum SUV (SUVmax) on 18F-FDG PET/CT in intervals of 10%. The results were expressed as L1.0-L5.0, and L10%-L50%, respectively. The pathological length of gross tumor (Lpath) was calculated based on the shrinkage ratio of primary tumor. The measurable lymph nodes were measured on PET/CT preoperatively, labeled during operation, and examined for pathology. RESULTS: Lpath was 6.87 ± 2.25 cm, L30% and L2.5 were 6.61 ± 1.76 cm and 7.56 ± 1.89 cm, respectively. L30% was closer to Lpath than other % SUVmax, L2.5 was closer to Lpath than other absolute SUV thresholds. The diagnostic performance of 18F-FDG PET/CT for lymph nodes was best at the cutoff SUV of 2.7, providing sensitivity of 70% and a specificity of 83.7% for detecting lymph node metastases. CONCLUSIONS: The tumor length with 30% SUVmax as the threshold was closest to the actual pathological length of EGJC. The diagnostic efficiency of 18F-FDG PET/CT was best at the cutoff SUVmax of 2.7 for detecting lymph node metastases in EGJC.

Hispidulin inhibits hepatocellular carcinoma growth and metastasis through AMPK and ERK signaling mediated activation of PPARγ.

Hispidulin, a phenolic flavonoid, exerts potent cytotoxicity towards a variety of human cancers. However, the effects of hispidulin on hepatocellular carcinoma (HCC) and underlying molecular mechanisms of its action remain elusive. The present study investigated the effect of hispidulin on HCC in experimental models, including tumor cell lines and mouse tumor xenograft. Results demonstrated that hispidulin was cytotoxic and anti-proliferative to HCC cell lines (SMMC7721 and Bel7402). Hispidulin activated caspase-3 and triggered apoptosis in HCC cells. Moreover, hispidulin inhibited cell migration and invasion by inhibiting the expression of matrix metalloproteinases (MMP-2, MMP-9) and by inducing tissue inhibitor of metalloproteinase-3 (TIMP-3) expression. Hispidulin activated peroxisome proliferator-activated receptor γ (PPARγ) signaling which mainly contributed to its cytotoxicity in HCC cells. Remarkably, GW9662 (a PPARγ inhibitor) or PPARγ targeting siRNA significantly abrogated the anti-proliferative, pro-apoptotic, and anti-metastatic effects of hispidulin in HCC cells. Furthermore, hispidulin induced activation of PPARγ which was associated with increased phosphorylation of AMPK, ERK, JNK in HCC cells. Compound C (an AMPK inhibitor) or PD98059 (a MEK inhibitor) partly reversed the effects of hispidulin on PPARγ signaling in HCC cells. In contrast, no significant changes in PPARγ signaling were observed in HCC cells pretreated with SP600125 (a JNK inhibitor), while SP6000125 significantly inhibited the anti-cancer effects of hispidulin in HCC cells. Hispidulin administration effectively suppressed Bel7402 xenograft tumor growth and lung metastasis in vivo. Our findings indicate that PPARγ activation by hispidulin effectively suppressed HCC cell growth and metastasis both in vitro and in vivo.

What Is the Appropriate Clinical Target Volume for Esophageal Squamous Cell Carcinoma? Debate and Consensus Based on Pathological and Clinical Outcomes.

Accurate delineation of clinical target volume (CTV) is critical in the effective management of squamous cell carcinoma (SCC) of esophagus using radiation therapy. Accurate delineation may improve the probability of local control and reduce the risk of complications. However, there are no consistent standards on the proper size of the margins added to the gross tumor volume (GTV). Different institutions and radiation oncologists have discordant opinions. In this paper, we review pathological and clinical outcomes to determine the most appropriate CTV for squamous cell carcinomas (SCC) of esophagus. The CTV for esophageal carcinoma should ensure that all subclinical lesions are encompassed regardless of the physical distance. The most precise method for delineating a reasonable CTV is to combine advanced imaging techniques, such as PET/CT and EUS, which allows the detection and prediction of subclinical lesions based on tumor characteristics such as the pathological type, differentiation, T disease, length and lymph node status.

Distributions of angiogenesis and lymphangiogenesis in gastrointestinal intramucosal tumors.

BACKGROUND: Although angiogenesis and lymphangiogenesis in gastrointestinal cancers has been investigated in many studies, their distribution characteristics in gastrointestinal intramucosal tumors have not been well addressed. METHODS: We evaluated the blood microvessel density (BMVD) and lymphatic microvessel density (LMVD) by immunostaining with monoclonal antibodies of CD34 and D2-40 in 37 patients with stomach intramucosal carcinoma and 28 patients with colorectal intramucosal neoplasia. Microvessels with endothelial cells labeled by CD34 but not by D2-40 were recognized as blood microvessels; and microvessels with endothelial cells labeled by both CD34 and D2-40 were recognized as lymphatic vessels. Furthermore, the relationships between expression of vascular endothelial growth factor (VEGF), VEGF-C, and BMVD, LMVD were investigated as well. RESULTS: The LMVD was significantly higher in peritumoral tissues than in corresponding normal tissues in gastrointestinal intramucosal tumors (20.87 versus 14.56, P = 0.003). However, there was no significant difference in the BMVD between peritumoral tissues and corresponding normal tissues (P = 0.166). The BMVD in peritumoral tissues was higher in patients with lymph node metastases than in patients without lymph nodes metastases (P = 0.047). Our results did not show significant association between VEGF, VEGF-C and BMVD, LMVD. CONCLUSIONS: Our results suggested that the increase of lymphangiogenesis seems superior to the increase of angiogenesis in gastrointestinal intramucosal tumors.

Reversal MDR in breast carcinoma cells by transfection of ribozyme designed according the secondary structure of mdr1 mRNA.

Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. The present study aims to investigate whether the ribozyme could reverse MDR in breast carcinoma cells. In this study, two GUC sites (GUC106 and GUC135) on the surface of mdr1 mRNA were selected according to the secondary structure of the 5'-region of mdrl mRNA. The ribozyme gene RZ106 and RZ135 complementary to two sides bases of the target GUC were synthesized and cloned into the plasmid pEGFP -C1 which has EGFP (Enhanced Green Fluorescence Protein) as report gene and Kan/Neo as selection gene. After transfection with the recombinant plasmid and selected by G418, the stable cell clones were produced and used for detection. The alteration of mdr1 mRNA and P-gp in the treated cells was detected by RT-PCR, flow cytometry and Rh123 retention. The reversal efficiency of the drug resistance for adriamycin was determined by MTT assay. The results showed that after transfection with RZ106 and RZ135, the amount of the mdr1 mRNA and P-gp decreased significantly and the efflux function of P-gp was inhibited accordingly. Nine-fold and 16-fold reduction of resistance for adriamycin was observed in the two groups of treated cells. These results suggested that both ribozymes can reverse the MDR phenotype by inhibiting the expression of mdr1 mRNA and P-gp, and the RZ135 showed the better cleavage efficiency. The ribozyme strategy designed according the secondary structure of the target RNA could be a useful therapy for reversal of MDR.

Alteration of cyclin D1 in gastric carcinoma and its clinicopathologic significance.

AIM: To detect the genetic alteration and abnormal expression of cyclin D1 in gastric carcinoma and investigate its clinicopathologic significance in advanced gastric carcinoma. METHODS: Proteins of cyclin D1 were detected by immunohistochemistry in 42 cases of advanced gastric carcinoma with their follow-up data available, 27 cases of early stage carcinoma, 21 cases of gastric adenoma, 22 cases of hyperplastic polyp and 20 cases of normal mucosa adjacent to adenocarcinomas. Genetic alteration of cyclin D1 was detected by Southern blot and expression of cyclin D1 mRNA was detected by PT-PCR in 42 cases of advanced gastric carcinoma. RESULTS: Cyclin D1 protein was not expressed in normal mucosa, hyperplastic polyp and gastric adenoma, while it was only positively expressed in gastric carcinoma. The expression rate of cyclin D1 protein in early stage gastric carcinoma, advanced gastric carcinoma and lymph node metastasis was 48.1%, 47.4% and 50.0%, respectively. The amplification of cyclin D1 gene was detected in 16.6% of advanced gastric carcinomas. The overexpression of cyclin D1 mRNA was detected in 40.5% of the samples. There was no significant correlation between cyclin D1 protein expression and age, lymph-node metastasis and histological grading in patients with advanced gastric carcinoma (chi2 = 0.038, 0.059, 0.241, P>0.05). Significant correlation was observed between the expression of cyclin D1 protein and the 5-year survival rate (chi2 = 3.92, P<0.05). CONCLUSION: Detection of cyclin D1 protein by immunohistochemistry may be useful in the diagnosis of early gastric carcinomas. Patients with positive expression of cyclin D1 protein tend to have a worse prognosis.