Chris Zhiyi Zhang

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 (IC50 = 6.9) and CNE-2 (IC50 = 7.4) cells but minimally affected normal NP 69 cells. Further investigation disclosed that MCL induced apoptosis, DNA fragmentation, G1-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 G1-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. Cancer Prev Res; 5(1); 109–21. ©2011 AACR.

Dihydroartemisinin exhibits antitumor activity toward hepatocellular carcinoma in vitro and in vivo

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua L., has been shown to exhibit inhibitory effects on human cancer cells. However, its antitumor ability toward hepatocellular carcinoma (HCC) has not been studied. In this study, we demonstrated that DHA significantly inhibited HCC cell growth in vitro and in vivo via inducing G2/M cell cycle arrest and apoptosis. The induction of p21 and the inhibition of cyclin B and CDC25C contributed to DHA-induced G2/M arrest. DHA-induced apoptosis was associated with mitochondrial membrane depolarization, release of cytochrome c, activation of caspases, and DNA fragmentation. Activation of caspase 9 and caspase 3, but not caspase 8, was detected in DHA-treated cells. Attenuation of apoptosis in cells pretreated with Z-VAD-FMK suggested the involvement of caspase cascade. Furthermore, p53 facilitated apoptosis caused by DHA. Bcl-2 family proteins were also responsible for DHA-induced apoptosis. DHA exposure decreased Mcl-1 expression but increased the levels of Noxa and active Bak. Bak was released from the Mcl-1/Bak complex due to the decline of Mcl-1. Further study revealed that Mcl-1 was rapidly degraded in DHA-treated cells and that DHA-induced apoptosis was largely inhibited by overexpression of Mcl-1 or RNAi-mediated decrease of Bak and Noxa. In a HCC-xenograft mouse model, the intraperitoneal injection of DHA resulted in significant inhibition of HCC xenograft tumors. Taken together, our data, for the first time, demonstrate the potential antitumor activity of DHA in HCC.

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.

Low SIRT3 Expression Correlates with Poor Differentiation and Unfavorable Prognosis in Primary Hepatocellular Carcinoma

SIRT3, a mitochondrial sirtuin belonging to nicotinamide adenine nucleotide (NAD) dependent deacetylases, is implicated in metabolism, longevity and carcinogenesis. SIRT3 expression and its significance in hepatocellular carcinoma (HCC) remain largely unclear. In this study, we demonstrated that SIRT3 expression in HCC tissue was much lower than that in paracarcinoma tissue, at both mRNA and protein levels. The cutoff value for low SIRT3 expression in HCC was defined according to receiver operating characteristic curve (ROC) analysis. As disclosed by immunohistochemistry (IHC) results, low SIRT3 expression was present in 67.3% (167/248) of HCC cases. Furthermore, low expression of SIRT3 was significantly correlated to differentiation (P = 0.013), clinical stage (P = 0.005), serum AFP level (P<0.01), tumor multiplicity (P = 0.026) and relapse (P = 0.028). Moreover, Kaplan-Meier analysis indicated that low SIRT3 expression associated with unfavorable overall survival (P<0.01) and recurrence-free survival (P = 0.004). The prognostic impact of SIRT3 was further confirmed by stratified survival analysis. Importantly, multivariate analysis revealed that low SIRT3 expression was an independent poor prognostic marker for overall survival (Hazard Ratio (HR) 0.555, 95% confidence interval (95% CI) 0.344–0.897, P = 0.016). Collectively, we conclude that SIRT3 is decreased in HCC and is a novel unfavorable marker for prognosis of patients with this fatal disease.

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.

miR-625 suppresses tumour migration and invasion by targeting IGF2BP1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and the third leading cause of cancer-related deaths worldwide. Tumour metastasis is one of the major causes of high mortality. microRNAshave been implicated in HCC metastasis. In this study, we found that miR-625 was frequently downregulated in HCC samples. A decrease in miR-625 was significantly correlated with lymph node anddistance metastasis (P=0.013), the presence of portal venous invasion (P=0.036), tumor-node-metastasis (TNM) stage (P=0.027) and unfavourable overall survival (P=0.003). Compared with primary tumours, miR-625 expression was markedly reduced in portal venous metastatic tumours. Re-expression of miR-625 in HCC cells was remarkably effective in suppressing cell migration andinvasiveness in vitro and in vivo. Mechanistically, miR-625 was confirmed to downregulate IGF2 mRNA-binding protein 1(IGF2BP1) directly, the expression of which was inversely correlated with the level of miR-625 in HCC cell lines and tissues. High expression of IGF2BP1 was frequently found in HCC samples, and associated with poor prognosis. Knockdown of endogenous IGF2BP1 by siRNA exhibited similar effects as the overexpression of miR-625, whereas overexpression of IGF2BP1 (without the 3′-UTR) abrogated miR-625-mediated metastasis inhibition. Interference of the PTEN/HSP27 pathway contributed to miR-625-mediated metastasis inhibition. Taken together, our data suggest that miR-625 might function as an antimetastatic miRNA to have an important role in HCC progression by modulating the IGF2BP1/PTEN pathway. The newly identified miR-625/IGF2BP1 axis represents a new potential therapeutic target for HCC treatment.

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.

Downregulation of Polo-Like Kinase 4 in Hepatocellular Carcinoma Associates with Poor Prognosis

Polo-like kinase 4 (PLK4), belonging to serine/threonine kinase family, is critical for centriole replication and cell cycle progression. PLK4 has been proposed as a tumor suppressor in hepatocellular carcinoma (HCC). However, its expression and significance in HCC have not been well studied. In the present study, we found that PLK4 was markedly downregulated in both HCC cell lines and fresh cancer tissues, using quantitative real-time-PCR and western blot. Immunohistochemistry data also revealed that decreased expression of PLK4 was present in 72.4% (178/246) of HCC tissues, compared with the corresponding adjacent nontumorous tissues. Furthermore, PLK4 expression significantly correlated with clinicopathological parameters, including clinical stage (P = 0.034), serum α-fetoprotein (AFP) (P = 0.019) and tumor size (P = 0.032). Moreover, HCC patients with low PLK4 expression survived shorter than those with high PLK4 expression, as indicated by overall survival (P = 0.002) and disease-free survival (P = 0.012) assessed by the Kaplan–Meier method. In addition, multivariate analysis suggested PLK4 as an independent predictor of overall survival (HR, 0.556; 95%CI, 0.376−0.822; P = 0.003) and disease-free survival (HR, 0.547; 95%CI, 0.382−0.783; P = 0.001). Collectively, our study demonstrated that PLK4 was remarkably downregulated in HCC and could be served as a potential prognostic marker for patients with this deadly disease.

Transcription factor ZBP-89 in cancer growth and apoptosis.

ZBP-89, a Krüppel-type zinc-finger transcription factor that binds to GC-rich sequences, is involved in the regulation of cell growth and cell death. It maps to chromosome 3q21 and is composed of 794 residues. Having bifunctional regulatory domains, ZBP-89 may function as a transcriptional activator or repressor of variety of genes such as p16 and vimentin. ZBP-89 arrests cell proliferation through its interactions with p53 and p21(waf1). It is able to stabilize p53 through directly binding and enhance p53 transcriptional activity by retaining it in the nucleus. In addition, ZBP-89 potentiates in butyrate-induced endogenous p21(waf1) up-regulation. ZBP-89 is usually over-expressed in human cancer cells, where it can efficiently induce apoptosis through p53-dependent and -independent mechanisms. Moreover, ZBP-89 is capable of enhancing killing effects of several anti-cancer drugs. Therefore, ZBP-89 may be served as a potential target in cancer therapy.

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.