Anti-Cancer Agents in Medicinal Chemistry (v.17, #1)

Meet Our Editorial Board Member by Luigi Quintieri (1-1).

Overview by Michelle Prudhomme (2-2).

PREFACE by Michelle Prudhomme (3-3).

Thiol-Activated Anticancer Agents: The State of the Art by Daniele Dalzoppo, Veronica Di Paolo, Laura Calderan, Gianfranco Pasut, Antonio Rosato, Anna Maria Caccuri, Luigi Quintieri (4-20).
The thiol or sulfhydryl group, as part of low molecular weight non-peptide biomolecules, as well as part of the cysteine residues in peptides and proteins, is known to play extremely important roles in several aspects of cellular function. Glutathione (?-Glu-Cys-Gly; GSH) is the most abundant thiol-containing peptide in mammals, being present intracellularly in the low millimolar concentration range, but only in the low micromolar concentration range in the majority of extracellular fluids. Notably, intracellular levels of GSH have been found to be significantly upregulated in a number of human cancers, a phenomenon thought to contribute, in concert with overexpression of some GSHassociated enzymes, to the development of tumor cell chemo- and radioresistance. On the other hand, various natural and synthetic chemical entities of different sizes show significant cytotoxic activity only upon interaction with a thiol, and can therefore exploit the GSH-rich intracellular environment of tumors. This review article attempts to summarize the current structural and pharmacological knowledge in the field of thiol-activated anticancer agents, with a focus on the mechanism(s) of their activation. Even though a great part of the available thiol-activated anticancer compounds is still in the preclinical phase of testing, some of them are undergoing trials in cancer patients.

Anticancer Drug Combinations, How Far We can Go Through? by Da-Yong Lu, En-Hong Chen, Hong-Ying Wu, Ting-Ren Lu, Bin Xu, Jian Ding (21-28).
Many clinical cancer therapies are less effective by using one anticancer drug only due to refractory properties of cancer pathogenesis and drug resistance property in advanced cancer patients. A general consensus among clinicians is that anticancer drug cocktail might better control cancer progresses and metastasis than single drug therapeutics in clinical trials. Despite great popularity, the anticancer drug combination dogma has not been established. The complexity of drug combination dogma discovery is more than we can expect now. This article speculates possible routes we can undertake in this matter. The background knowledge of drug combination therapy presently practiced and possible future landscapes and drawbacks of cancer drug combinative therapies are highlighted.

Ruthenium(II) Complexes as Potential Apoptosis Inducers in Chemotherapy by Kangdi Zheng, Qiong Wu, Chengxi Wang, Weijun Tan, Wenjie Mei (29-39).
Herein, the development of ruthenium complexes as potential apoptosis inducers, as well as their underlying mechanism has been reviewed. In recent years, various ruthenium complexes have been designed and their in vitro and in vivo inhibitory activities against various types of tumor cells have been evaluated extensively. It's demonstrated that ruthenium complexes can induce apoptosis of tumor cells through the signal pathway of mitochondria-mediated, death receptor-mediated, and/or endoplasmic reticulum (ER) stress pathways. Alternately, the binding behavior of these ruthenium(II) complexes with DNA, especially with Gquadruplex DNA may play a key role in the DNA damage of tumor cells, and thus provides a versatile tool to rational design novel ruthenium complexes with high activity and selectivity.

Killing Glioma 'Stem-like' Cells via Drug-Induced Relocation of Endosomal Urokinase Proteins by Fredric A. Gorin, Nagarekha Pasupuleti, Dinesh Mahajan, Sundeep Dugar (40-47).
High grade gliomas (HGGs) are primary CNS cancers with more than 95% of patients experiencing tumor recurrence following radiation therapy, chemotherapy, and/or an anti-angiogenic therapy. Populations of glioma 'stem-like' cells (GSCs) exist in both proliferative and non-proliferative states and are capable of tumor regrowth. These GSCs survive within hypoxic tumor regions and avascular tumor margins, while retaining the capability to regenerate. Successful treatment of HGGs depends on therapeutic targeting of GSCs to avert tumor regeneration. Here, we review novel intracellular mechanisms by which 3-amino-5-arylamino-6-chloro-N-(diaminomethylene) pyrazine-2-carboximide (UCD38B) and the much more potent 5'-substituted arylamino compounds (cmpd 10357) irreversibly kill GSCs utilizing caspase-independent, programmed necrotic cell death. Drug-induced relocation of a subset of endosomes to perinuclear mitochondria triggers the mitochondrial release and nuclear translocation of apoptosis inducible factor (AIF) that is followed by nuclear condensation and cancer cell demise. This drug-induced endosomal 'mis-trafficking' affects a subset of endosomes containing proteins belonging to the urokinase plasminogen activator system (uPAS) and guided by lipoprotein receptor protein type 1 (LRP-1). UCD38B and congeners act intracellularly and bind to intracellular urokinase plasminogen activator (uPA) to disrupt uPA binding to PAI-1 and the endosomal LRP-1 guidance protein. These small molecules are cytotoxic to persistently hypoxic and acidotic HGG cell lines and to high grade gliomas from patient derived xenografts (PDX). Immunodeficient mice with intracerebral PDX glial tumors demonstrate drug-specific, AIF- mediated necrosis after 24h of treatment. The propensity of these small molecules to kill non-proliferating and proliferating hypoxic GSCs, suggests a potential synergistic therapeutic role with radiotherapy, anti-mitotic and anti-angiogenic therapies.

Rapamycin-loaded Immunoliposomes Functionalized with Trastuzumab: A Strategy to Enhance Cytotoxicity to HER2-positive Breast Cancer Cells by Josimar O. Eloy, Raquel Petrilli, Robert W. Brueggemeier, Juliana Maldonado Marchetti, Robert J. Lee (48-56).
Background: Liposomes have been employed to improve pharmacokinetics and reduce side effects of drugs. They can be functionalized with antibodies for targeted delivery. While the monoclonal antibody trastuzumab has been employed in the therapy of HER2-positive breast cancer, the resistance developed during treatment has been reported. Rapamycin could be used in combination with trastuzumab for improved therapeutic response.
Objective: In this study, we aimed to develop rapamycin-loaded liposomes and immunoliposomes with trastuzumab, characterize them and evaluate their in vitro cytotoxicity.
Method: Formulations were prepared by the thin film hydration method and immunoliposome was conjugated to antibody by covalent bond. Characterization involved particle size, polydispersity, zeta potential, encapsulation efficiency, functionalization efficiency, DSC and FTIR assays. Cell studies were conducted through the MTT assay.
Results: SPC:Chol:DSPE-PEG formulation prepared at 1:10 drug to lipid ratio presented high encapsulation efficiency, appropriate particle size, low polydispersity, negative zeta potential and colloidal stability. Rapamycin exhibited intermolecular interactions with lipids and underwent crystallinity reduction. Rapamycin-loaded immunoliposomes were prepared with high trastuzumab functionalization efficiency and antibody stability. Cytotoxicity studies showed that the HER2-positive SK-BR-3 cell line was sensitive to trastuzumab, either as free drug or in the context of immunoliposomes, and is more sensitive to rapamycin than the triple negative MDA-MB-231 cells. For MDA-MB-231, the liposomal rapamycin was more cytotoxic than the free drug. Furthermore, the immunoliposomes showed potent cytotoxicity against SK-BR-3 cells. Finally, rapamycin and trastuzumab exhibited in vitro synergistic effect, particularly through immunoliposomes.
Conclusion: The formulation developed herein has potential for in vivo evaluation.

Novel 2-Thienyl- and 2-Benzothienyl-Substituted 6-(2-Imidazolinyl)Benzothiazoles: Synthesis; in vitro Evaluation of Antitumor Effects and Assessment of Mitochondrial Toxicity by Livio Racané, Mirela Sedi|, NataŠa Ili|, Maja Aleksi|, Sandra Kraljevi| Paveli|, Grace Karminski-Zamola (57-66).
Background: Differently substituted thiophenes are largely studied due to their diverse pharmacological properties, especially anticancer activity. Recent studies have reported on interesting benzothiophene compounds antitumor properties and we also recently reported on the synthesis, strong antitumor activities and DNA binding features of substituted thieno[3',2':4,5]thieno- and benzo[b]thieno[2,3-c]quinolones, containing different substituents, mostly amidino- or substituted amidino- groups.
Objective: The objective of presented paper was to prepare a series of novel cationic 2-thiophene and 2- benzothiophene substituted 6-(2- imidazolinyl)benzothiazole derivatives and to test their antiproliferative activity against several human cancer cell lines.
Method: Synthesis of 2-thiophene and 2-benzothiophene substituted 6-(2-imidazolinyl)benzothiazole derivatives was carried out by condensation reaction of 2-amino-5-(2-imidazolinium)benzenethiolate with aldehydes or carbonyl chloride derivatives followed by two simple acid-base reaction steps was used for their conversion into targeted mesylates. Evaluation of antiproliferative effects and cell death was done by use of MTT assay and annexin-V test, while expression of sphingosine kinase 1 was studied by Western blot and gluthatione intracellular levels were measured by use of a luminescence-based assay.
Results: Preparation of water soluble mesylate salts 3a-3j was successfully achieved. In general, all compounds showed pronounced anticancer activities in vitro. Compound 3f showed strong and selective cytostatic activity in cervical carcinoma cells (HeLa) with moderate toxicity on normal fibroblasts. Similar to all other tested compounds, 3f cannot be considered a mitochondrial toxicant. One of the major mechanisms accounting for observed cytostatic effects of 3f was induction of apoptosis, probably due to specific inhibition of acid ceramidase activity. Compound 3h negatively regulated activity of sphingosine kinase 1 in HeLa cells.
Conclusion: Design of novel inhibitors targeting enzymes that regulate sphingolipid biosynthesis and turnover could change the landscape for the development of new anticancer drugs.

Design, Synthesis and Evaluation of 5-pyridin-4-yl-2-thioxo-[1,3,4]oxadiazol-3-yl Derivatives as Anti-angiogenic Agents Targeting VEGFR-2 by Umesh Bhanushali, Samidha Kalekar-Joshi, Renuka Kulkarni-Munshi, Swapna Yellanki, Raghavender Medishetty, Pushkar Kulkarni, Ramaa Subramanian Chelakara (67-74).
Background: Angiogenesis is physiological process in embryogenesis, organ development, endometrial vasculature in menstrual cycle and wound healing. Angiogenesis has also been associated with several pathological conditions such as cancer, arthritis, atherosclerosis, etc. Out of the many growth factor responsible for angiogenesis, vascular endothelial growth factor (VEGF) is one of the most important and positive regulator of angiogenesis with its distinct specificity for vascular endothelial cells. The current work is the small efforts towards development of newer inhibitor of angiogenesis targeting VEGFR-2.

Objective: With the view to develop inhibitors of angiogenesis, pharmacophore characteristics were used to design aromatic/ heteroaromatic ring containing compounds. These compounds were then docked in to the active side of VEGFR-2 with the aid of docking. They were then synthesize and spectrally characterized and carry out in-vitro and in-vivo anti-angiogenic evaluation studies to ascertain its angiogenesis inhibition potential.
Result: 3-substituted-5-(4-pyridin-4yl)-1,3,4-oxadiazole-2-thiones designed as inhibitors of angiogenesis targeting VEGFR2. In docking study, all the molecules showed similar way of binding with VEGFR2 as that of the cocrystallised ligand. Compound 3i and 3j were found to be most active in the series showing good inhibition of angiogenesis in both CAM and in zebrafish embryo assays. The compound 3i was the most active in the series with IC50 of 0.5 ?M for VEGR-2.
Conclusion: To conclude the work we have successfully designed newer inhibitors of angiogenesis targeting VEGFR- 2. These compounds were then screen and found to inhibit angiogenesis of CAM and zebrafish at dose of 1 ?M.

Synthesis, in vitro Antiproliferative and Antiinflammatory Activities, and Kinase Inhibitory effects of New 1,3,4-triarylpyrazole Derivatives by , Mohammed I. El-Gamal, Mohammed S. Abdel-Maksoud, Mahmoud M. Gamal El-Din, Ji-Sun Shin, Kyung-Tae Lee, Kyung Ho Yoo, Chang-Hyun Oh (75-84).
Background: Pyrazole derivatives have been reported as both anticancer and antiinflammatory agents.
Objective: This study was conducted to develop new pyrazole derivatives as potential anticancer and/or antiinflammatory agents. Their molecular mechanisms of action have been investigated.
Method: a series of new triarylpyrazole derivatives were synthesized. Their in vitro anticancer activity was tested against NCI-58 cancer cell line panel of nine cancer types. The most active compound 1a was tested against sixteen kinases, many of them are known to be over-expressed in leukemia and breast cancer. The most sensitive kinases were V600E-B-RAF, C-RAF, FLT3, and P38?/MAPK14. Compound 1a was further tested for caspase-3/7 activity and LDH release assay as measures of its apoptotic and necrotic activities against RPMI-8226. Moreover, the ability of compounds 1a, 1b, and 1g to inhibit nitric oxide and prostaglandin E2production in LPS-induced RAW 264.7 macrophages was also examined.
Results: Compounds 1a, 1c, and 1g showed the highest activities against the cancer cell line panel, with more inhibitory effects against leukemia and breast cancer subpanels. The highest activity was exerted by compound 1a. Its IC50 values against RPMI-8226, K-562 leukemia cell lines, and MDA-MB-468 breast cancer cell line were 1.71 ?M, 3.42 ?M, and 6.70 ?M, respectively. The IC50 of compound 1a against P38?/MAPK14 kinase was 0.515 ?M. The caspase activity was increased by 72% and 170% at 1.23 ?M and 3.70 ?M concentrations of compound 1a, respectively. Furthermore, compound 1b inhibited 80.26% and 95.31% of NO and PGE2 productions, respectively, at 50 ?M concentration in the LPS-induced RAW 264.7 macrophages.
Conclusion: Compound 1a could kill the cells through induction of apoptosis rather than necrosis. Compound 1a was more selective against cancer cells than non-cancerous cells. In addition, the hydroxyl analogue 1b was the most active as antiinflammatory agent.

New 2-Imino-2H-Chromene-3(N-aryl)carboxamides as Potential Cytotoxic Agents by Rupinder Kaur Gill, Jyoti Kumari, Jitender Bariwal (85-92).
Synthesis and structure activity relationships of four series of novel 2-imino-2H-chromene-3(N-aryl) carboxamides (V-VIII) have been described by bioisosteric replacement of usually present ketone at 2nd position of coumarin with imine. Various substitutents are introduced on aryl and chromene ring of iminocoumarin to investigate the effect of lipophilicity and electronic properties of substituents on cytotoxic activity against four human cancer cell lines. Novel 2-imino-2H-chromene-3(N-aryl)carboxamides (V-VIII) were synthesized by the reaction of substituted 2- cyanoacetamides with different salicyaldehydes in the presence of sodium acetate in glacial acetic acid. Compound VIa showed potent activity against MCF-7 (IC50 = 8.5 ?M), PC-3 (IC50 = 35.0 ?M), A-549 (IC50 = 0.9 ?M) and Caco-2 (IC50 = 9.9 ?M) cell lines. The anticancer results revealed that most of the synthesized compounds showed equipotent activity with the standard 5-fluorouracil and docetaxel on Caco-2 and MCF-7 cell lines, respectively

Effect of PUFAs Oral Administration on the Amount of Apoptotic Caspases Enzymes in Gastric Cancer Patients Undergoing Chemotherapy by Homayun Dolatkhah, Ahmad Movahedian, Mohammad-Hossein Somi, Mahmud Aghaei, Naser Samadi, Ahmad Mirza-Aghazade, Ali Esfahani (93-101).
Objective: Gastric cancer is the fourth most common cancer and the second cause of death in the world. According to the studies, the gastric cancer is relatively sensitive to chemotherapy. The aim of this study was to investigate the association of oral administer PUFAs with Caspase enzymes in patients with gastric cancer under chemotherapy.
Methods: This study was a Clinical Trial in which the target group consisted of the patients recognized with gastric cancer for the first time and cured under chemotherapy. Thirty-four patients were selected and categorized randomly into two groups. The case group included the patients taking PUFAs along with chemotherapeutic agent. In these patients, chemotherapy started with Cis-Platin plus PUFAs supplement in the scale of 3600 mg daily and in three courses. In control group, the individuals were under the same chemotherapy protocol without PUFAs. Biopsy samples from tumor were taken from the patients before and after chemotherapy. Levels of mRNA and protein expression of caspase 3, 8, 9 were measured in biopsy samples by Real-Time PCR and Frozen Section methods. The levels of apoptosis were determined using DNA-damage colorimetric assay.
Results: In the case group, caspase 3 showed a significant increase in both gene and protein expression levels after administration of PUFAs supplement in comparison with those of the control group (p=0.006 for gene, p=0.001 for protein). PUFAs induced caspase-9 gene expression level in these patients (p<0.0001). Caspase-9 protein level also revealed a marked elevation when PUFAs were administered along with chemotherapeutic agent (p<0.0001). DNA damage in gastric tissue from the patients under PUFAs treatment plus Cis-Platin was significantly higher than that of control group (p=0.003). PUFAs showed no significant changes in caspase-8 both at the gene and protein levels in the patients.
Conclusion: According to the results of present study, it appears that oral administration of PUFAs can elevate the efficacy of chemotherapy agent in individuals' mitochondria-dependent apoptosis. As PUFAs enhances caspase-3 and 9 genes expression levels, which is an important induce the mitochondrial dependent apoptosis process.
The study was registered in Iran clinical trials registry center under No. IRCT2014031016922N1.

Glycodendrimer PPI as a Potential Drug in Chronic Lymphocytic Leukaemia. The Influence of Glycodendrimer on Apoptosis in In Vitro B-CLL Cells Defined by Microarrays by Ida Franiak-Pietryga, Ewelina Zi&#243;|kowska, Barbara Ziemba, Dietmar Appelhans, Henryk Maciejewski, Brigitte Voit, Aleksandra Kaczmarek, Tadeusz Robak, Barbara Klajnert-Maculewicz, Barbara Cebula-Obrzut, Piotr Smolewski, Maciej Borowiec, Maria Bryszewska (102-114).
Background: Chronic lymphocytic leukaemia (CLL) cells are characterized by failures in the apoptosis pathway and increased proliferation, resulting in the progressive accumulation of B-lymphocytes in blood. Despite the wide range of antileukaemic drugs, CLL remains an incurable disease. However, a breakthrough is expected which will allow more effective treatment.
Objective: The study investigates the influence of poly(propyleneimine) (PPI) dendrimer with peripheral amino groups, 30% of which were coated with maltotriose (PPI-G4-OS-Mal-III), on CLL cells, and demonstrates that it acts through the induction of the apoptotic mechanism. It is important to note that the dendrimer was used as a drug itself and not as a drug carrier.
Method: CLL and normal lymphocytes were treated in vitro with the dendrimer, either alone or in combination with fludarabine (FA). The percentages of apoptotic and necrotic cells, and the protein expression, were checked using a flow cytometer. Gene expression was screened using a two-colour microarray with 60-mer probes.
Results: The results confirm that PPI-G4-OS-Mal-III influences the viability of CLL cells in vitro and does not exert any significant harmful effect on normal lymphocytes. The dendrimer appears to significantly influence gene and protein expression in CLL cells.
Conclusion: Since dendrimers can be specifically targeted, they may be very effective in CLL therapy, especially since in vitro PPI-G4-OS-Mal-III has been found to have stronger effect than fludarabine.

Background: Intrinsic resistance to apoptotic cell death due to co-occurrence of mutated KRAS and p53 is frequently reported in pancreatic cancer that renders them aggressive, highly proliferative and metastatic. In addition, these cancer types are less sensitive to apoptosis inducing drugs where promotion of autophagic cell death could be a viable option for treatment under such circumstances.
Objective: In this study we examined the potential of three intrinsically fluorescent benzo[?]phenoxazines or BPZs (R=Cl, CH3, H) to induce cytotoxic autophagy in chemo and apoptosis-resistant, KRAS and p53 mutated pancreatic cancer model cell line, MIAPaCa-2.
Methods: Cells were adapted at in vitro metabolically stressed condition (5% serum) to initiate intrinsic cell survival strategies within. Cell proliferation, colonogenicity, cellular uptake, retention, localization, cellular granularity and presence of both apoptosis and autophagy biomarkers were assessed in BPZ treated/untreated (solvent) cells to validate induction of concentration dependent cytotoxic autophagy and other consequences.
Results: For the first time, we report the ability of this class of compounds to accumulate within cells increasing its granularity, inducing death via autophagy. From different kinetics study, it was observed that the autophagic-cell death was dependent on the ligand type, duration of incubation or working concentrations.
Conclusion: Among the three BPZ tested, both 3B (benzo[?]phenoxazine-5-one) and 2B (10-methyl-benzo[?] phenoxazine-5-one) induced pro-death autophagy in MIAPaCa-2 cells at an IC50 of 5 ?M and 20 ?M respectively. Such compounds would be of great interest to explore as novel cytotoxic autophagy inducing agents in apoptosisresistant cancer types.

In Vivo Performance of a Ruthenium-cyclopentadienyl Compound in an Orthotopic Triple Negative Breast Cancer Model by Nuno Mendes, Francisco Tortosa, Andreia Valente, Fernanda Marques, Ant&#243;nio Matos, T&#226;nia S. Morais, Ana Isabel Tomaz, F&#225;tima G&#228;rtner, M. Helena Garcia (126-136).
Background: Ruthenium-based anti-cancer compounds are proposed as viable alternatives that might circumvent the disadvantages of platinum-based drugs, the only metallodrugs in clinical use for chemotherapy. Organometallic complexes in particular hold great potential as alternative therapeutic agents since their cytotoxicity involves different modes of action and present reduced toxicity profiles.
Objective: During the last few years our research group has been reporting on a series of organometallic ruthenium(II)- cyclopentadienyl complexes with important cytotoxicity against several cancer cell lines, surpassing cisplatin in activity. We report herein preliminary in vivo studies with one representative compound of this family, with exceptional activity against several human cancer cell lines, including the glycolytic and highly metastatic MDAMB231 cell line used in this study.
Method: The anti-tumor activity of our compound was studied in vivo on N:NIH(S)II-nu/nu nude female mice bearing triple negative breast cancer (TNBC) orthotopic tumors. Administration of 2.5 mg/kg/day during ten days caused cell death mostly by necrosis (in vitro and in vivo), inducing tumor growth suppression of about 50% in treated animals when compared to controls.
Results: The most remarkable result supporting the effectiveness and potential of this drug was the absence of metastases in the main organs of treated animals, while metastases were present in the lungs of all control mice, as revealed by histopathological and immunohistochemical analysis.
Conclusion: These in vivo studies suggest a dual effect for our drug not only by suppressing growth at the primary tumor tissue but also by inhibiting its metastatic behavior. Altogether, these results represent a benchmark and a solid starting point for future studies.

Background: Prenyl flavonoid icaritin (1) and ?-anhydroicaritin (2) are two natural products with important biological and pharmacological effects. such as antiosteoporosis, estrogen regulation and antitumor properties.
Objective: The present study investigates the synthesis and cytotoxic activities on three Human cancer cell lines (Hela, HCC1954 and SK-OV-3) of icaritin and β-anhydroicaritin Mannich base derivatives in vitro models.
Method: Preylated flavonoid icaritin (1) upon treatment with formic acid under microwave assistance gave another natural product β-anhydroicaritin (2) in good yield (89%). Based on Mannich reaction of 1 or 2 with various secondary amines and formaldehyde, two series eighteen new 6-aminomethylated flavonoids Mannich base derivatives 3-11 and 12-20 were synthesized. Their cytotoxic potential against three human cancer cell lines (Hela, HCC1954 and SK-OV-3) were evaluated by the standard MTT method with cis-Platin and Paclitaxel as positive control.
Results: Our research showed that most of these flavonoid Mannich base derivatives displayed equal or higher (lower IC50 values) cytotoxic activities than the positive control cis-Platin. Some compounds possess the IC50 value below 10µM. Compounds 6-(diisopropylamino)methyl- and 6-morpholinylmethyl substituted β-anhydroicaritin (15 and 19) showed selective cytotoxicity against HCC1954 cells (IC50 12.688 µM) and Hela cells (IC50 6.543 µM) respectively.
Conclusion: Our finding most of icaritin and β-anhydroicaritin Mannich base derivatives possessing moderate to potent cytotoxicity against these three cancer cells (Hela, HCC1954 and SK-OV-3). Compound 15 and 19 showed selective cytotoxicity against HCC1954 cells and Hela cells respectively, they are potential and selective anticancer agent and worthy of further development.

Cordycepin Affects Multiple Apoptotic Pathways to Mediate Hepatocellular Carcinoma Cell Death by Yulin Zhou, Zhihua Guo, Qingfan Meng, Jiahui Lu, Ning Wang, Hui Liu, Qiming Liang, Yutong Quan, Di Wang, Jing Xie (143-149).
Background: Cordycepin possesses anti-inflammatory, anti-metastatic and anti-tumor properties.
Objective: The present study investigates the anti-hepatocellular carcinoma activities of cordycepin in in vitro and in vivo models.
Method: Cell viability, apoptosis rate, intracellular reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) were determined by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide bromide assay, annexin V/propidium iodide double staining, 2',7'-dichlorfluorescein-diacetate and 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining respectively. The expressions of pro-apoptosis and antiapoptosis proteins were detected by western blot. A PLC/PRL/5-xenografted nude mouse model was applied to further confirm the anti-tumor activities of cordycepin.
Results: Cordycepin suppressed cell viability, enhanced apoptotic rate, inhibited cell proliferation and increased cleaved poly (ADP-ribose) polymerase (PARP) level. Apoptotic alteration on mitochondria and abnormal changes on b-cell lymphoma 2 (Bcl-2) and b-cell lymphoma-extra large (Bcl-xL) levels were observed in cordycepin-treated cells. Furthermore, cordycepin suppressed the activation of extracellular signaling-regulated kinase (ERKs) and mammalian target of rapamycin (mTOR) in both PLC/PRF/5 and HepG2 cells. Finally, PLC/PRL/5-xengrafted BALB/c athymic nude mice were performed to confirm cordycepin's anti-tumor action.
Conclusion: Our finding suggests that the anti-hepatocellular carcinoma properties of cordycepin are related to its modulation of multiple anti-apoptotic and pro-apoptotic pathways. Our study provides an experimental evidence for cordycepin as a rational agent for hepatocellular carcinoma treatment.