Correction: Zheng, L., et al. PBN11-8, a Cytotoxic Polypeptide Purified from Marine Bacillus, Suppresses Invasion and Migration of Human Hepatocellular Carcinoma Cells by Targeting Focal Adhesion Kinase Pathways. Polymers 2018, 10, 1043.

The authors wish to make a change to the published paper [...].

Exosomes-coated bcl-2 siRNA inhibits the growth of digestive system tumors both in vitro and in vivo.

BACKGROUND: The therapeutic application of small interfering RNA is limited by the lack of an effective delivery system to tumors. In the present study, an efficient approach to deliver siRNA to cancer cells using exosome system was developed. MATERIALS & METHODS: Exosomes were isolated from bovine milk and exosomes-coated bcl-2 siRNA (exosiBcl-2) was synthesized using ultrasonic approach. The anticancer effect of exsosiBcl-2 was studied by Confocal microscopy, flow cytometry, scratch wound healing and Transwell experiments, RT-qPCR and Western blot approaches, etc. Xenograft nude mice tumor model was performed to check the antitumor activity of exosiBcl-2 in vivo. RESULTS: ExosiBcl-2 can cross the cell membrane efficiently and inhibit the growth of cancer cells. ExosiBcl-2 treatment led to apoptosis and dramatic inhibition of migration and invasion of cancer cells via downregulating metastatic related genes. In vivo study revealed that exosiBcl-2 inhibited the tumor growth significantly in nude mice. CONCLUSION: ExosiBcl-2 has potential to be developed as a novel anticancer agent.

Comparison of the Interactions of Different Growth Factors and Glycosaminoglycans.

Most growth factors are naturally occurring proteins, which are signaling molecules implicated in cellular multiple functions such as proliferation, migration and differentiation under patho/physiological conditions by interacting with cell surface receptors and other ligands in the extracellular microenvironment. Many of the growth factors are heparin-binding proteins (HBPs) that have a high affinity for cell surface heparan sulfate proteoglycans (HSPG). In the present study, we report the binding kinetics and affinity of heparin interacting with different growth factors, including fibroblast growth factor (FGF) 2,7,10, hepatocyte growth factor (HGF) and transforming growth factor (TGF ß-1), using a heparin chip. Surface plasmon resonance studies revealed that all the tested growth factors bind to heparin with high affinity (with KD ranging from ~0.1 to 59 nM) and all the interactions are oligosaccharide size dependent except those involving TGF ß-1. These heparin-binding growth factors also interact with other glycosaminoglycans (GAGs), as well as various chemically modified heparins. Other GAGs, including heparan sulfate, chondroitin sulfates A, B, C, D, E and keratan sulfate, showed different inhibition activities for the growth factor-heparin interactions. FGF2, FGF7, FGF10 and HGF bind heparin but the 2-O-sulfo and 6-O-sulfo groups on heparin have less impact on these interactions than do the N-sulfo groups. All the three sulfo groups (N-, 2-O and 6-O) on heparin are important for TGFß-1-heparin interaction.

Recent Progress of Marine Polypeptides as Anticancer Agents.

BACKGROUND: Marine environment constitutes an almost infinite resource for novel anticancer drug discovery. The biodiversity of marine organisms provides a rich source for the discovery and development of novel anticancer peptides in the treatment of human cancer. Marine peptides represent a new opportunity to obtain lead compounds in biomedical field, particularly for cancer therapy. OBJECTIVE: Providing an insight of the recent progress of patented marine peptides and presenting information about the structures and mechanistic mode of anticancer activities of these marine peptides. METHODS: We reviewed recent progress on the patented anticancer peptides from marine organisms according to their targets on different signal pathways. This work focuses on relevant recent patents (2010-2018) that entail the anticancer activity with associated mechanism and related molecular diversity of marine peptides. The related cellular signaling pathways for novel peptides that induce apoptosis and affect tubulin-microtubule equilibrium, angiogenesis and kinase activity that are related to the anticancer and related pharmacological properties are also discussed. RESULTS: The recent patents (2010-2018) of marine peptides with anticancer activity were reviewed, and the anticancer activity of marine peptides with associated mechanism and related molecular diversity of marine peptides were also discussed. CONCLUSION: Marine peptides possess chemical diversity and display potent anticancer activity via targeting different signal pathways. Some of the marine peptides are promising to be developed as novel anticancer agents.

Molybdenum oxide quantum dots prepared via a one-step stirring strategy and their application as fluorescent probes for pyrophosphate sensing and efficient antibacterial materials.

MoOx quantum dots (QDs) were prepared using a one-step stirring treatment of molybdenum trioxide (MoO3) powder in dimethyl sulfoxide (DMSO). Besides their highly sensitive label-free metal ion and pyrophosphate sensing, the antibacterial activity of the as-prepared MoOx QDs was discovered for the first time.

PBN11-8, a Cytotoxic Polypeptide Purified from Marine Bacillus, Suppresses Invasion and Migration of Human Hepatocellular Carcinoma Cells by Targeting Focal Adhesion Kinase Pathways.

The development of antitumor drugs has attracted cancer researchers and the identification of novel antitumor lead compounds is certainly of great interest. The fermentation broth of Bacillus sp. N11-8, which was isolated from the Antarctic waters, showed cytotoxicity towards different cells. A cytotoxic polypeptide, PBN11-8, was purified from the fermentation broth of Bacillus sp. N11-8 using ultrafiltration, ammonium sulfate precipitation, anion exchange liquid chromatography and high performance liquid chromatography (HPLC). Cloning and sequence analysis showed that PBN11-8 polypeptide (MW: ~19 kDa by the electrospray-ionization (ESI)) displayed high similarity with peptidase M84 from Bacillus pumilus. PBN11-8 possessed moderate cytotoxicity towards several cancer cell lines with IC50 values of 1.56, 1.80, 1.57, and 1.73 µg/mL against human hepatocellular carcinoma cell line BEL-7402, human renal clear cell adenocarcinoma cell line 786-0, human hepatocellular carcinoma cell line HepG2, and human pancreatic cancer cell line Panc-28, respectively. Moreover, the polypeptide displayed weak cytotoxicity towards normal cell line renal tubular epithelial cell line HK2 and human normal liver cell line L02 cells. Wound healing migration and Transwell experiments demonstrate that PBN11-8 could inhibit the migration and invasion of BEL-7402. Further investigation revealed that PBN11-8 suppresses focal adhesion kinase (FAK)-mediated adhesion, migration, and invasion by disturbing FAK/extracellular regulated protein kinases (ERK) signaling and matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9 (MMP-9) in BEL-7402 cells. Thus, PBN11-8 represents a potential novel anti-cancer lead compound.

Screening of microorganisms from Antarctic surface water and cytotoxicity metabolites from Antarctic microorganisms.

The Antarctic is a potentially important library of microbial resources and new bioactive substances. In this study, microorganisms were isolated from surface water samples collected from different sites of the Antarctic. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay-based cytotoxicity-tracking method was used to identify Antarctic marine microorganism resources for antitumor lead compounds. The results showed that a total of 129 Antarctic microorganism strains were isolated. Twelve strains showed potent cytotoxic activities, among which a Gram-negative, rod-shaped bacterium, designated as N11-8 was further studied. Phylogenetic analysis based on 16S rRNA gene sequence showed that N11-8 belongs to the genus Bacillus. Fermented active products of N11-8 with molecular weights of 1-30 kDa had higher inhibitory effects on different cancaer cells, such as BEL-7402 human hepatocellular carcinoma cells, U251 human glioma cells, RKO human colon carcinoma cells, A549 human lung carcinoma cells, and MCF-7 human breast carcinoma cells. However, they displayed lower cytotoxicity against HFL1 human normal fibroblast lung cells. However, they displayed lower cytotoxicity against HFL1 human normal fibroblast lung cells. Microscopic observations showed that the fermented active products have inhibitory activity on BEL-7402 cells similar to that of mitomycin C. Further studies indicated that the fermented active products have high pH and high thermal stability. In conclusion, most strains isolated in this study may be developed as promising sources for the discovery of antitumor bioactive substances. The fermented active products of Antarctic marine Bacillus sp. N11- 8 are expected to be applied in the prevention and treatment of cancer.

ERK inhibition sensitizes cancer cells to oleanolic acid-induced apoptosis through ERK/Nrf2/ROS pathway.

Oleanolic acid (OA) is a natural triterpenoid that is widely distributed in edible and medicinal plants. OA exerts anti-tumor activity on a wide range of cancer cells primarily through inducing apoptosis. Dysregulated ERK signaling is closely complicated in the biology of cancer, such as metastasis, proliferation, and survival, and it can be activated by various stimuli. In this study, we found that OA induced the activation of ERK in cancer cells. ERK activation compromised the apoptosis induced by OA. Blocking ERK activation by U0126 or siRNAs was able to potentiate the pro-apoptotic activity of OA on cancer cells. OA was shown to promote ERK-dependent Nrf2 expression in cancer cells, and in turn, Nrf2 expression was able to suppress OA-induced ROS generation. Blockade of Nrf2 expression was able to increase ROS levels and apoptotic death in cancer cells. In conclusion, we provided evidences that ERK activation is a mechanism underlying the resistance of cancer cells to OA-induced apoptosis and targeting ERK is a promising strategy to enhance the anti-tumor efficacy of OA.

Isolation and characterization of marine Brevibacillus sp. S-1 collected from South China Sea and a novel antitumor peptide produced by the strain.

A Gram-positive, rod-shaped bacterium, designated as S-1, was isolated from a marine sediment sample collected from South China Sea. Phylogenetic analysis based on 16S rRNA gene sequence showed that S-1 belongs to the genus Brevibacillus. A novel cytotoxic peptide was isolated from the fermentation broth of the marine-derived bacterium Brevibacillus sp. S-1, using ion-exchange chromatography and reverse-phase HPLC chromatography. The molecular weight of this peptide was determined as 1570 Da by MALDI-TOF mass spectrometry, and its structure was proposed as a cyclic peptide elucidated by MALDI-TOF/TOF mass spectrometry and de novo sequencing. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay showed that this peptide exhibited cytotoxicity against BEL-7402 human hepatocellular carcinoma cells, RKO human colon carcinoma cells, A549 human lung carcinoma cells, U251 human glioma cells and MCF-7 human breast carcinoma cells. Additionally, SBP exhibited low cytotoxicity against HFL1 human normal fibroblast lung cells. The result suggested that the cytotoxic effect of the peptide is specific to tumor cells.

Aplysin sensitizes cancer cells to TRAIL by suppressing P38 MAPK/survivin pathway.

TNF-related apoptosis-inducing ligand (TRAIL) is a tumor-selective apoptosis inducer and has been shown to be promising for treating various types of cancers. However, the application of TRAIL is greatly impeded by the resistance of cancer cells to its action. Studies show that overexpression of some critical pro-survival proteins, such as survivin, is responsible for TRAIL resistance. In this study, we found that Aplysin, a brominated compound from marine organisms, was able to restore the sensitivity of cancer cells to TRAIL both in vitro and in vivo. Aplysin was found to enhance the tumor-suppressing capacity of TRAIL on several TRAIL-resistant cancer cell lines. TRAIL-induced apoptosis was also potentiated in A549 and MCF7 cells treated with Aplysin. Survivin downregulation was identified as a mechanism by which Aplysin-mediated TRAIL sensitization of cancer cells. Furthermore, the activation of p38 MAPK was revealed in Aplysin-treated cancer cells, and its inhibitor SB203580 was able to abrogate the promoting effect of Aplysin on the response of cancer cells to TRAIL action, as evidenced by restored survivin expression, elevated cell survival and reduced apoptotic rates. In conclusion, we provided evidence that Aplysin acts as a sensitizer for TRAIL and its effect on p38 MAPK/survivin pathway may partially account for this activity. Considering its low cytotoxicity to normal cells, Aplysin may be a promising agent for cancer treatment in combination with TRAIL.

Oleanolic acid inhibits proliferation and invasiveness of Kras-transformed cells via autophagy.

Oleanolic acid (OA) has been widely studied because of its pleiotropic therapeutic and preventive effect on various diseases. However, the mechanisms of OA's action are still not clear yet, especially its suppressing effect on transformed cells. In this work, we found that OA induced autophagy in normal tissue-derived cells without cytotoxicity. OA-induced autophagy was shown to decrease the proliferation of KRAS-transformed normal cells and to impair their invasion and anchorage-independent growth. Interrupting autophagy rescued OA's effect on the transformed cells. Mouse model experiments also demonstrated that OA suppressed the growth of KRAS-transformed breast epithelial cell MCF10A-derived tumor xenograft by inducing autophagy. Finally, we identified that OA induced autophagy in normal cells by inhibiting the activation of Akt/mTOR/S6K signaling. In conclusions, we found that OA treatment permitted normal cells to undergo autophagy. The induced autophagy was required for OA to prevent or delay the growth of transformed normal cells.

p38 MAPK signaling mediates mitochondrial apoptosis in cancer cells induced by oleanolic acid.

Oleanolic acid (OA) is a nutritional component widely distributed in various vegetables. Although it has been well recognized for decades that OA exerts certain anti-tumor activity by inducing mitochondria-dependent apoptosis, it is still unclear that what molecular signaling is responsible for this effect. In this study, we employed cancer cell lines, A549, BXPC-3, PANC-1 and U2OS to elucidate the molecular mechanisms underlying OA anti- tumor activity. We found that activation of MAPK pathways, including p-38 MAPK, JNK and ERK, was triggered by OA in both a dose and time-dependent fashion in all the tested cancer cells. Activation was accompanied by cleavage of caspases and PARP as well as cytochrome C release. SB203580 (p38 MAPK inhibitor), but not SP600125 (JNK inhibitor) and U0126 (ERK inhibitor), rescued the pro-apoptotic effect of OA on A549 and BXPC- 3 cells. OA induced p38 MAPK activation promoted mitochondrial translocation of Bax and Bim, and inhibited Bcl-2 function by enhancing their phosphorylation. OA can induce reactive oxygen species (ROS)-dependent ASK1 activation, and this event was indispensable for p38 MAPK-dependent apoptosis in cancer cells. In vivo, p38 MAPK knockdown A549 tumors proved resistant to the growth-inhibitory effect of OA. Collectively, we elucidated that activation of ROS/ASK1/p38 MAPK pathways is responsible for the apoptosis stimulated by OA in cancer cells. Our finding can contribute to a better understanding of molecular mechanisms underlying the antitumor activity of nutritional components.

Oleanolic acid induces protective autophagy in cancer cells through the JNK and mTOR pathways.

Autophagy is a biological process that eliminates damaged or excessive proteins and is utilized by various types of cells to maintain cellular homeostasis. Autophagy also occurs in cancer cells and exerts anti-survival or pro-survival effects depending on stimuli, nutrient and context. Oleanolic acid (OA), a widely spread natural compound, induces apoptosis in a range of cancer cells. However, some tumor cell lines are resistant to the pro-apoptotic effect of OA, and the mechanism remains unknown. In the present study, we found that OA induced autophagic event in cancer cells in a dose- and time-dependent manner, evidenced by an increased ratio between LC3-II and LC3-I and frequent granulation of LC3 proteins in OA-stimulated tumor cell lines. Inhibition of autophagy potentiated the pro-apoptotic activity of OA on cancer cells. Furthermore, the JNK and mTOR signaling pathways were found to be affected by OA treatment. Interfering with JNK and mTOR abolished OA-induced autophagy and sensitized cancer cells to apoptosis. Collectively, we showed that OA was able to initiate protective autophagy, which compromised the antitumor activity of OA on cancer cells. Blocking autophagy may be a promising strategy to enhance the tumor suppressor activity of OA.

Oleanolic acid induces metabolic adaptation in cancer cells by activating the AMP-activated protein kinase pathway.

Cancer cells are well-known to require a constant supply of protein, lipid, RNA, and DNA via altered metabolism for accelerated cell proliferation. Targeting metabolic pathways is, therefore, a promising therapeutic strategy for cancers. Oleanolic acid (OA) is widely distributed in dietary and medicinal plants and displays a selective cytotoxicity to cancer cells, primarily by inducing apoptosis and cell cycle arrest. This study investigated if OA inhibited growth of tumor cells by affecting their metabolism. OA was found to activate AMP-activated protein kinase (AMPK), the master regulator of metabolism, in prostate cancer cell line PC-3 and breast cancer cell line MCF-7. AMPK activation is required for the antitumor activity of OA on cancer cells. Lipogenesis, protein synthesis, and aerobic glycolysis were inhibited in cancer cells treated with OA, in an AMPK activation-dependent manner. The metabolic alteration was shown to mediate the tumor suppressor activity of OA on cancer cells. Collectively, this study provides evidence that OA, as a widely distributed nutritional component, is able to exert antitumor function by interfering in the metabolic pathway in cancer cells. This finding should encourage researchers to study if affecting cancer metabolism is a common mechanism by which nutritional compounds suppress cancers.

CS5931, a novel polypeptide in Ciona savignyi, represses angiogenesis via inhibiting vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs).

CS5931 is a novel polypeptide from Ciona savignyi with anticancer activities. Previous study in our laboratory has shown that CS5931 can induce cell death via mitochondrial apoptotic pathway. In the present study, we found that the polypeptide could inhibit angiogenesis both in vitro and in vivo. CS5931 inhibited the proliferation, migration and formation of capillary-like structures of HUVECs (Human Umbilical Vein Endothelial Cell) in a dose-dependent manner. Additionally, CS5931 repressed spontaneous angiogenesis of the zebrafish vessels. Further studies showed that CS5931 also blocked vascular endothelial growth factor (VEGF) production but without any effect on its mRNA expression. Moreover, CS5931 reduced the expression of matrix metalloproteinases (MMP-2 and MMP-9) both on protein and mRNA levels in HUVEC cells. We demonstrated that CS5931 possessed strong anti-angiogenic activity both in vitro and in vivo, possible via VEGF and MMPs. This study indicates that CS5931 has the potential to be developed as a novel therapeutic agent as an inhibitor of angiogenesis for the treatment of cancer.

Cloning, characterization and expression of a cDNA encoding a granulin-like polypeptide in Ciona savignyi.

Previous study in our laboratory confirmed that a novel polypeptide, CS5931 derived from Ciona savignyi possesses potent antitumor activity. In the present study, the full length cDNA of CS5931 precursor, termed Cs-pgrn-1 was cloned. The complete cDNA sequence of this gene consists of 685 bp containing an open reading frame (ORF) of 522 bp (173 amino acid residues). In silico analysis revealed that the polypeptide consists of two identical domains, similar with granulin (GRN) found in other species, and each of the domain encodes a polypeptide identical with CS5931. Phylogenetic analysis confirmed that CS5931 shares high homology with Ciona intestinalis GRN and is conserved during evolution. The polypeptide also shows high similarity with human GRN A, B, and C. Prediction of 3D protein structure revealed the 3D structure of CS5931 is very similar with human GRN A. The CS5931 was expressed using a prokaryotic expression system and the purified polypeptide inhibited the growth of several tumor cell lines in vitro via apoptotic pathway. Our study revealed that CS5931 has the potential to be developed as a novel antitumor agent.

Targeting cellular apoptotic pathway with peptides from marine organisms.

Apoptosis is a critical defense mechanism against the formation and progression of cancer and exhibits distinct morphological and biochemical traits. Targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. Peptides from marine organisms have become important sources in the discovery of antitumor drugs, especially when modern technology makes it more and more feasible to collect organisms from seas. This primer summarizes several marine peptides, based on their effects on apoptotic signaling pathways, although most of these peptides have not yet been studied in depth for their mechanisms of action. Novel peptides that induce an apoptosis signal pathway are presented in association with their pharmacological properties.

A novel polypeptide from Meretrix meretrix Linnaeus inhibits the growth of human lung adenocarcinoma.

A novel polypeptide (Mere15) was purified from Meretrix meretrix Linnaeus by ammonium sulfate fractionation, ion exchange, gel filtration and reversed phase chromatography. Mere15 exhibited selective cytotoxicity to several human cancer cells. In vivo study showed that Mere15 significantly suppressed the growth of human lung adenocarcinoma A549 xenograft in nude mice. The mechanism was associated with a G(2)/M phase arrest followed by apoptosis, including membrane blebbing, loss of mitochondrial membrane potential, externalization of phosphatidylserine, chromosome condensation and DNA fragmentation. Western blot analysis showed that the intrinsic pathway was involved in Mere15-induced apoptosis. These results suggest that Mere15 may have therapeutic potential for the treatment of non-small-cell lung carcinoma.

A novel polypeptide extracted from Ciona savignyi induces apoptosis through a mitochondrial-mediated pathway in human colorectal carcinoma cells.

BACKGROUND: Sessile marine animals such as sponges, ascidians, and bryozoans are a rich source of bioactive natural products, many of which exhibit potent anticancer activity. MATERIALS AND METHODS: We extracted and purified a polypeptide with potent antitumor activity from Ciona savignyi by acetone fractionation, ultrafiltration, ion exchange chromatography, gel chromatography, and high-performance liquid chromatography. An MTT assay was used to study the cytotoxicity of the isolated fraction and the purified polypeptide. Cell cycle and Western blot analysis were performed to study the mode of action of the purified polypeptide. RESULTS: A novel polypeptide with potent antitumor activity was purified. The molecular weight of the polypeptide, designated CS5931, was 5931 Da, and use of the genome basic local alignment search tool (BLAST) revealed that the N-terminal sequence of CS5931 is identical to that of granulin A from C savignyi. CS5931 exhibited significant cytotoxicity for several cancer cell types and induced apoptotic death in HCT-8 cells in a dose- and time-dependent manner. Cell cycle analysis demonstrated that CS5931 caused cell cycle arrest at the G(2)/M phase, and a sub-G(1) peak appeared after treating the cells with CS5931 for 12 hours. The mitochondrial-mediated pathway was implicated in CS5931-induced apoptosis. CONCLUSION: Our observations clearly demonstrate the antiproliferative and proapoptotic activities of the polypeptide CS5931 from C savignyi and the mitochondrial-mediated pathway involved in the polypeptide-induced cell death.

Antitumor peptides from marine organisms.

The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new antitumor agents in the field of the development of marine bioactive substances. In this review, the progress on studies of antitumor peptides from marine sources is provided. The biological properties and mechanisms of action of different marine peptides are described; information about their molecular diversity is also presented. Novel peptides that induce apoptosis signal pathway, affect the tubulin-microtubule equilibrium and inhibit angiogenesis are presented in association with their pharmacological properties. It is intended to provide useful information for further research in the fields of marine antitumor peptides.