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.

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.

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.

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.