Clinicopathologic features, tumor immune microenvironment and genomic landscape of Epstein-Barr virus-associated intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Little is known about Epstein-Barr virus (EBV)-associated intrahepatic cholangiocarcinoma (EBVaICC) because of its rarity. We aimed to comprehensively investigate the clinicopathology, tumor immune microenvironment (TIME) and genomic landscape of this entity in southern China. METHODS: We evaluated 303 intrahepatic cholangiocarcinomas (ICCs) using in situ hybridization for EBV. We compared clinicopathological parameters between EBVaICC and nonEBVaICC, and we analyzed EBV infection status, tumor-infiltrating lymphocytes (TILs) and genomic features of EBVaICC by immunohistochemistry, double staining, nested PCR, multiplex immunofluorescence staining, fluorescence in situ hybridization and whole-exome sequencing. RESULTS: EBVaICC accounted for 6.6% of ICCs and was associated with EBV latency type I infection and clonal EBV isolates. Patients with EBVaICC were more often female and younger, with solitary tumors, higher HBV infection rates and less frequent cirrhosis; the lymphoepithelioma-like (LEL) subtype was more common in EBVaICC. EBVaICC was associated with a significantly larger TIME component than nonEBVaICC. The LEL subtype of EBVaICC - associated with a significantly increased density and proportion of CD20+ B cells and CD8+ T cells - was associated with significantly higher 2-year survival rates than conventional EBVaICC and nonEBVaICC. Both PD-1 and PD-L1 in TILs, and PD-L1 in tumor cells, were overexpressed in EBVaICC. High PD-L1 expression in tumor cells and high CD8+ TIL densities were significantly more common in EBVaICC than in nonEBVaICC. Seven genes (MUC4, DNAH1, GLI2, LIPE, MYH7, RP11-766F14.2 and WDR36) were mutated in at least 3 patients. EBVaICC had a different mutational pattern to liver fluke-associated cholangiocarcinoma and HBV-associated ICC. CONCLUSIONS: EBVaICC, as a subset of ICC, has unique etiological, clinicopathological and genetic characteristics, with a significantly larger TIME component. Paradoxically, patients with EBVaICC could be candidates for immune checkpoint therapy. LAY SUMMARY: Epstein-Barr virus (EBV) is associated with a subtype of intrahepatic cholangiocarcinoma, with unique clinicopathological and genetic characteristics. The tumor immune microenvironment is also different in this tumor subtype and patients with EBV-associated intrahepatic cholangiocarcinoma may respond well to immune checkpoint inhibitors.

High-expression of ZBP-89 correlates with distal metastasis and poor prognosis of patients in clear cell renal cell carcinoma.

ZBP-89, a Krüppel-type zinc-finger transcription factor, is found to participate in tumor development, invasion and metastasis. However, the expression status of ZBP-89 in clear cell renal cell carcinoma (CCRCC) remains elusive. Using quantitative real-time-PCR and Western Blot, we found that, in fresh cancer tissues, ZBP-89 was remarkably decreased in 79.2% (19/24) and 83.3% (5/6) of CCRCC at mRNA and protein level, respectively. Immunohistochemistry also revealed a significant decline of ZBP-89 expression in CCRCC, showing that low expression of ZBP-89 was present in 73.9% (105/142) of tumorous tissues but in 48.1% (52/108) of the corresponding adjacent kidney tissues. Furthermore, ZBP-89 expression in CCRCC was significantly correlated with several clinicopathological features, including TNM stage (P=0.005) and distal metastasis (P=0.001). Further study confirmed that ZBP-89 expression was markedly higher in metastatic CCRCC than that in non-metastatic tissue (P=0.002). In addition, CCRCC patients with low ZBP-89 expression survived longer than those with high ZBP-89 expression, as indicated by the result of univariate analysis (P<0.0001). More importantly, multivariate analysis revealed that ZBP-89 was an independent predictor of overall survival (HR, 2.871; 95% CI, 1.409-5.853; P=0.004). Collectively, our study provides vigorous evidence that ZBP-89 was significantly downregulated in CCRCC and could be served as a promising biomarker for prediction of distal metastasis and prognosis of patient with CCRCC.

Trichosanthin inhibits breast cancer cell proliferation in both cell lines and nude mice by promotion of apoptosis.

Breast cancer ranks as a common and severe neoplasia in women with increasing incidence as well as high risk of metastasis and relapse. Translational and laboratory-based clinical investigations of new/novel drugs are in progress. Medicinal plants are rich sources of biologically active natural products for drug development. The 27-kDa trichosanthin (TCS) is a ribosome inactivating protein purified from tubers of the Chinese herbal plant Trichosanthes kirilowii Maximowicz (common name Tian Hua Fen). In this study, we extended the potential medicinal applications of TCS from HIV, ferticide, hydatidiform moles, invasive moles, to breast cancer. We found that TCS manifested anti-proliferative and apoptosis-inducing activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cells. Flow cytometric analysis disclosed that TCS induced cell cycle arrest. Further studies revealed that TCS-induced tumor cell apoptosis was attributed to activation of both caspase-8 and caspase-9 regulated pathways. The subsequent events including caspase-3 activation, and increased PARP cleavage. With regard to cell morphology, stereotypical apoptotic features were observed. Moreover, in comparison with control, TCS- treated nude mice bearing MDA-MB-231 xenograft tumors exhibited significantly reduced tumor volume and tumor weight, due to the potent effect of TCS on tumor cell apoptosis as determined by the increase of caspase-3 activation, PARP cleavage, and DNA fragmentation using immunohistochemistry. Considering the clinical efficacy and relative safety of TCS on other human diseases, this work opens up new therapeutic avenues for patients with estrogen-dependent and/or estrogen-independent breast cancers.

Decreased expression of zinc-alpha2-glycoprotein in hepatocellular carcinoma associates with poor prognosis.

BACKGROUND: Zinc-alpha2-glycoprotein (AZGP1, ZAG) was recently demonstrated to be an important factor in tumor carcinogenesis. However, AZGP1 expression in hepatocellular carcinoma (HCC) and its significance remain largely unknown. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to determine mRNA level of AZGP1 in 20 paired fresh HCC tissues. Clinical and pathological data of 246 HCC patients were collected. Tissue-microarray-based immunohistochemistry (IHC) was performed to examine AZGP1 expression in HCC samples. Relationship between AZGP1 expression and clinicopathological features was analyzed by Chi-square test, Kaplan-Meier analysis and Cox proportional hazards regression model. RESULTS: AZGP1 expression was significantly lower in 80.0% (16/20) of tumorous tissues than that in the corresponding adjacent nontumorous liver tissues (P < 0.001). Consistently, IHC data revealed that decreased expression of AZGP1 was present in 80.1% (197/246) of HCC patient tissues (P < 0.001). Furthermore, AZGP1 expression in HCC significantly associated with several clinicopathological parameters, including serum AFP level (P = 0.013), liver cirrhosis (P = 0.002) and tumor differentiation (P = 0.025). Moreover, HCC patients with high AZGP1 expression survived longer, with better overall survival (P = 0.006) and disease-free survival (P = 0.025). In addition, low AZGP1 expression associated with worse relapse-free survival (P = 0.046) and distant metastatic progression-free survival (P = 0.036). CONCLUSION: AZGP1 was downregulated in HCC and could be served as a promising prognostic marker for HCC patients.

The MAP30 protein from bitter gourd (Momordica charantia) seeds promotes apoptosis in liver cancer cells in vitro and in vivo.

Human hepatocellular carcinoma Hep G2 cells and Hep G2-bearing mice were used as in vitro and in vivo models to assess the efficacy and safety of MAP30, a natural component from Momordica charantia, as an anticancer agent against liver cancer. Molecular studies disclosed the contribution of both caspase-8 regulated extrinsic and caspase-9 regulated intrinsic caspase cascades in MAP30-induced cell apoptosis. The antitumor potential was also effective in Hep G2-bearing nude mice. Since bitter gourd is a staple in many Asian countries, MAP30 would serve as a novel and relatively safe agent for prophylaxis and treatment of liver cancer.

In vitro and in vivo anticarcinogenic effects of RNase MC2, a ribonuclease isolated from dietary bitter gourd, toward human liver cancer cells.

Hepatocellular carcinoma (HCC) constitutes a predominant part of primary liver cancer which ranks as the fifth most common cancer as well as the third most common cause of cancer mortality. In view of the poor prognosis of unresectable liver cancers, it is of pivotal importance to develop novel chemotherapeutical regimens. RNase MC2 is a 14-kDa ribonuclease isolated from dietary bitter gourd (Momordica charantia) that manifested antitumor potential against breast cancers. In this study, we investigated the potential application of RNase MC2 on Hep G2 cells. We showed that RNase MC2 inhibited cell proliferation and induced cell apoptosis in both in vitro and in vivo studies. RNase MC2 treatment caused cell cycle arrest predominantly at the S-phase and apoptosis, which is associated with the activation of both caspase-8 and caspase-9 regulated caspase pathways. Our further investigation disclosed that RNase MC2 down-regulated the anti-apoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bak. Moreover, the phosphorylation of ERK and JNK was involved in the apoptosis process. Importantly, RNase MC2 significantly suppressed the growth of Hep G2 xenograft-bearing nude mice by inducing apoptosis. This notion is supported by data indicating an increased number of caspase-3- and PARP-positive cells, and TUNEL-positive cells in RNase MC2-treated tumor tissues. In summary, we have revealed the antitumor potential of RNase MC2 toward Hep G2 cells. Considering that bitter gourd is a common dietary component in many countries, this study may help to prompt the clinical application of RNase MC2.

RNase MC2: a new Momordica charantia ribonuclease that induces apoptosis in breast cancer cells associated with activation of MAPKs and induction of caspase pathways.

Ribonucleases (RNases) are ubiquitously distributed nucleases that cleave RNA into smaller pieces. They are promising drugs for different cancers based on their concrete antitumor activities in vitro and in vivo. Here we report for the first time purification and characterization of a 14-kDa RNase, designated as RNase MC2, in the seeds of bitter gourd (Momordica charantia). RNase MC2 manifested potent RNA-cleavage activity toward baker's yeast tRNA, tumor cell rRNA, and an absolute specificity for uridine. RNase MC2 demonstrated both cytostatic and cytotoxic activities against MCF-7 breast cancer cells. Treatment of MCF-7 cells with RNase MC2 caused nuclear damage (karyorrhexis, chromatin condensation, and DNA fragmentation), ultimately resulting in early/late apoptosis. Further molecular studies unveiled that RNase MC2 induced differential activation of MAPKs (p38, JNK and ERK) and Akt. On the other hand, RNase MC2 exposure activated caspase-8, caspase-9, caspase-7, increased the production of Bak and cleaved PARP, which in turn contributed to the apoptotic response. In conclusion, RNase MC2 is a potential agent which can be exploited in the worldwide fight against breast cancer.

Momordica Charantia lectin, a type II ribosome inactivating protein, exhibits antitumor activity toward human nasopharyngeal carcinoma cells in vitro and in vivo.

The incidence of nasopharyngeal carcinoma (NPC) remains high in endemic regions, including southern China, northern Africa, and North America. One of the promising therapeutic approaches on NPC is drug screening from natural products, such as components from traditional Chinese medicine. In this study, the antitumor activity of Momordica charantia lectin (MCL), a type II ribosome inactivating protein from bitter gourd, on NPC was investigated. MCL evinced potent cytotoxicity toward NPC CNE-1 (IC(50) = 6.9) and CNE-2 (IC(50) = 7.4) cells but minimally affected normal NP 69 cells. Further investigation disclosed that MCL induced apoptosis, DNA fragmentation, G(1)-phase arrest, and mitochondrial injury in both types of NPC cells. The reduction of cyclin D1 and phosphoretinoblastoma (Rb) protein expression contributed to arrest at G(1)-phase of the cell cycle. These events were associated with regulation of mitogen-activated protein kinases (MAPK; including p38 MAPK, JNK, and ERK) phosphorylation and promoted downstream nitric oxide (NO) production. Concurrent administration of the p38 MAPK inhibitor SB-203580 significantly diminished NO production and lethality of MCL toward NPC cells. Further studies revealed that MCL increased cytochrome c release into the cytosol, activated caspases-8, -9, and -3, and enhanced production of cleaved PARP, subsequently leading to DNA fragmentation and apoptosis. Finally, an intraperitoneal injection of MCL (1.0 mg/kg/d) led to an average of 45% remission of NPC xenograft tumors subcutaneously inoculated in nude mice. This is the first article that unveils the potential of a type II RIP, MCL, for prevention and therapy of NPC.