Intravenous injection of nattokinase-heparin electrostatic complex improves the therapeutic effect of advanced tumors by dissolving cancer-related thrombosis.

AIMS: Cancer-related thrombosis (CAT) is a common complication in cancer patients, significantly impacting their quality of life and survival prospects. Nattokinase (NK) has potent thrombolytic properties, however, its efficacy is limited by low oral bioavailability and the risk of severe allergic reactions with intravenous use. Heparin (HP) is a widely used anticoagulant in clinical settings. This study aimed to overcome the intravenous toxicity of NK and explore its effect on CAT in advanced tumors. MAIN METHODS: In this study, NK-HP electrostatic complexes were constructed, and their safety and thrombolytic efficacy were verified through guinea pig allergy tests, mouse tail vein tests, and both in vivo and in vitro thrombolysis experiments. Additionally, an S180 advanced tumor model was developed and combined with sialic acid-modified doxorubicin liposomes (DOX-SAL) to investigate the impact of NK-HP on CAT and its antitumor effects in advanced tumors. KEY FINDINGS: We observed that NK-HP can eliminate the intravenous injection toxicity of NK, has strong thrombolytic performance, and can prevent thrombosis formation. Intravenous injection of NK-HP can enhance the antitumor effect of DOX-SAL by reducing the fibrin content in advanced tumors and increasing the levels of the cross-linked protein degradation product D-dimer. SIGNIFICANCE: This study developed a method to eliminate the intravenous injection toxicity of NK, proposing a promising therapeutic strategy for CAT treatment, particularly for CAT in advanced tumors, and improving the efficacy of nano-formulations in anti-tumor therapy.

Vitamin K: Infection, Inflammation, and Auto-Immunity.

Vitamin K (VK) comprises a group of substances with chlorophyll quinone bioactivity and exists in nature in the form of VK1 and VK2. As its initial recognition originated from the ability to promote blood coagulation, it is known as the coagulation vitamin. However, based on extensive research, VK has shown potential for the prevention and treatment of various diseases. Studies demonstrating the beneficial effects of VK on immunity, antioxidant capacity, intestinal microbiota regulation, epithelial development, and bone protection have drawn growing interest in recent years. This review article focuses on the mechanism of action of VK and its potential preventive and therapeutic effects on infections (eg, asthma, COVID-19), inflammation (eg, in type 2 diabetes mellitus, Alzheimer's disease, Parkinson's disease, cancer, aging, atherosclerosis) and autoimmune disorders (eg, inflammatory bowel disease, type 1 diabetes mellitus, multiple sclerosis, rheumatoid arthritis). In addition, VK-dependent proteins (VKDPs) are another crucial mechanism by which VK exerts anti-inflammatory and immunomodulatory effects. This review explores the potential role of VK in preventing aging, combating neurological abnormalities, and treating diseases such as cancer and diabetes. Although current research appoints VK as a therapeutic tool for practical clinical applications in infections, inflammation, and autoimmune diseases, future research is necessary to elucidate the mechanism of action in more detail and overcome current limitations.

Role of emerging vitamin K­dependent proteins: Growth arrest­specific protein 6, Gla­rich protein and periostin (Review).

Vitamin K­dependent proteins (VKDPs) are a group of proteins that need vitamin K to conduct carboxylation. Thus far, scholars have identified a total of 17 VKDPs in the human body. In this review, we summarize three important emerging VKDPs: Growth arrest­specific protein 6 (Gas 6), Gla­rich protein (GRP) and periostin in terms of their functions in physiological and pathological conditions. As examples, carboxylated Gas 6 and GRP effectively protect blood vessels from calcification, Gas 6 protects from acute kidney injury and is involved in chronic kidney disease, GRP contributes to bone homeostasis and delays the progression of osteoarthritis, and periostin is involved in all phases of fracture healing and assists myocardial regeneration in the early stages of myocardial infarction. However, periostin participates in the progression of cardiac fibrosis, idiopathic pulmonary fibrosis and airway remodeling of asthma. In addition, we discuss the relationship between vitamin K, VKDPs and cancer, and particularly the carboxylation state of VKDPs in cancer.

Alternol/Alteronol: Potent Anti-cancer Compounds With Multiple Mechanistic Actions.

Alternol and its oxidate isomer Alteronol are small compounds isolated from the fermentation of a mutant fungus obtained from Taxus brevifolia bark. Preclinical studies showed their potent anti-cancer activities, including attenuating cellular survival pathways, altering protein levels of cell cycle regulators, activating xanthine dehydrogenase to cause accumulation of cellular reactive oxygen species and disrupting cell metabolism by disturbing four Krebs cycle enzymes specifically in malignant cells while having no significant effect on benign cells. In cancer cell culture models, Alternol or Alteronol exert their anti-cancer effect by inducing cell cycle arrest and triggering apoptotic cell death. In mice xenograft models, Alternol or Alteronol potently suppresses tumor growth with no obvious toxicity to the host with a wide therapeutic index over 30-fold. In conclusion, Alternol or Alteronol possess a great potential and feasibility to be developed as an effective anti-tumor therapeutic.

Development of a nattokinase-polysialic acid complex for advanced tumor treatment.

Cancer-associated thrombus (CAT) impedes delivery of nanoparticles to tumor sites and also inhibits the ability of immune cells to detect and attack these tumors, particularly in advanced tumors with old thrombi. Nattokinase (NK) is an extract from a popular Japanese food, natto, which consists of boiled soybeans fermented with bacteria. Nattokinase exerts strong fibrinolytic and thrombolytic activities and can unblock blood vessels. To deliver NK to thrombus sites in tumors, we modified the surface of NK with polysialic acid (PSA), which formed complexes via electrostatic interactions, resulting in NK-PSA. Particle size and zeta potential of NK-PSA were evaluated, and differential scanning calorimetry, Fourier-transform infrared spectroscopy, and morphological analyses of NK-PSA were performed. To determine the efficacy of the NK-PSA complex on delivery of nanoparticulate drugs, sialic acid-modified doxorubicin liposomes (DOX-SAL) were used as a model drug. In vivo pharmacokinetic and tissue distribution analyses showed that the blood clearance rate of DOX-SAL was significantly enhanced by NK-PSA, and NK-PSA increased accumulation of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) labeled SAL (DiR-SAL) in tumors. Analysis of anti-tumor efficacy showed that the combination of NK-PSA and DOX-SAL enhanced anti-tumor activity. These results suggested that NK-PSA combined with DOX-SAL may be an effective strategy to clear CAT and increase the ability of nanoparticles and immune cells to reach tumors.

Research progress on the anticancer effects of vitamin K2.

Despite the availability of multiple therapeutic methods for patients with cancer, the long-term prognosis is not satisfactory in a number of different cancer types. Vitamin K2 (VK2), which exerts anticancer effects on a number of cancer cell lines, is considered to be a prospective novel agent for the treatment of cancer. The present review aims to summarize the results of studies in which VK2 was administered either to patients with cancer or animals inoculated with cancerous cells, particularly investigating the inhibitory effects of VK2 on cancerous cells, primarily involving cell-cycle arrest, cell differentiation, apoptosis, autophagy and invasion. The present review summarizes evidence stating that treatment with VK2 could positively inhibit the growth of cancer cells, making it a potentially useful approach for the prevention and clinical treatment of cancer. Additionally, the combination treatment of VK2 and established chemotherapeutics may achieve better results, with fewer side effects. Therefore, more attention should be paid to the effects of micronutrients on tumors.

Evaluation of the antitumor effects of vitamin K2 (menaquinone-7) nanoemulsions modified with sialic acid-cholesterol conjugate.

Numerous studies have recently shown that vitamin K2 (VK2) has antitumor effects in a variety of tumor cells, but there are few reports demonstrating antitumor effects of VK2 in vivo. The antitumor effects of VK2 in nanoemulsions are currently not known. Therefore, we sought to characterize the antitumor potential of VK2 nanoemulsions in S180 tumor cells in the present study. Furthermore, a ligand conjugate sialic acid-cholesterol, with enhanced affinity towards the membrane receptors overexpressed in tumors, was anchored on the surface of the nanoemulsions to increase VK2 distribution to the tumor tissue. VK2 was encapsulated in oil-in-water nanoemulsions, and the physical and chemical stability of the nanoemulsions were characterized during storage at 25 °C. At 25 °C, all nanoemulsions remained physically and chemically stable with little change in particle size. An in vivo study using syngeneic mice with subcutaneously established S180 tumors demonstrated that intravenous or intragastric administration of VK2 nanoemulsions significantly suppressed the tumor growth. The VK2 nanoemulsions modified with sialic acid-cholesterol conjugate showed higher tumor growth suppression than the VK2 nanoemulsions, while neither of them exhibited signs of drug toxicity. In summary, VK2 exerted effective antitumor effects in vivo, and VK2 nanoemulsions modified with sialic acid-cholesterol conjugate enhanced the antitumor activity, suggesting that these VK2 may be promising agents for the prevention or treatment of tumor in patients.

Alternol, a natural compound, exerts an anti-tumour effect on osteosarcoma by modulating of STAT3 and ROS/MAPK signalling pathways.

Osteosarcoma (OS) is the most frequent primary malignant bone tumour. Alternol, a novel compound purified from microbial fermentation products exerts anti-tumour effects across several cancer types. The effect of alternol on human OS remains to be elucidated. We first evaluated the anti-tumour effect of alternol in several human OS cell lines in vitro and investigated its underlying mechanism. Alternol inhibited OS cell proliferation, migration and induced caspase-dependent apoptosis, G2/M cell cycle arrest in a dose and time-dependent manner. Moreover, alternol treatment inhibited signal transducer and activator of transcription-3 (STAT3) phosphorylation in 143B and MG63 human OS cells, as evaluated using a STAT3-dependent dual luciferase reporter system. Exposure to alternol resulted in excessive reactive oxygen species (ROS) generation and Jun amino-terminal kinases (JNK), extracellular signal-regulated kinases (ERK1/2) and p38 activation. Furthermore, alternol-induced cell death was significantly restored in the presence of the ROS scavenger, N-acetyl-l-cysteine (NAC) or a caspase inhibitor Z-VAD-FMK. NAC also prevented G2/M phase arrest and phosphorylation of mitogen-activated protein kinases (MAPK), but did not reverse STAT3 inactivation. Finally, alternol suppressed tumour growth in vivo in the nude mouse OS tibia orthotopic model. Immunohistochemistry revealed that alternol treatment resulted in down-regulation of phosph-STAT3 Tyr705 and up-regulation of cleaved caspase-3 and phosph-SAPK (Stress-activated protein kinases)/JNK expression. Taken together, our results reveal that alternol suppresses cell proliferation, migration and induces apoptosis, cell cycle arrest by modulating of ROS-dependent MAPK and STAT3 signalling pathways in human OS cells. Therefore, alternol is a promising candidate for developing anti-tumour drugs target OS.

Growth inhibition and apoptosis induction by alternol in pancreatic carcinoma cells.

AIM: To investigate the effect of alternol on pancreatic cancer cells. METHODS: Pancreatic cancer cells PANC-1 and BxPC3 were treated with various concentrations of alternol for 24, 48 and 72 h. Cell proliferation was measured by cell counting. Cell cycle distribution and mitochondrial membrane potential were determined by flow cytometry. Apoptosis was determined by a TdT-mediated dUTP nick end labeling assay and Hoechst staining. Expression of caspase 3, Bcl-2, p53 and p21 was measured by western blotting. RESULTS: Alternol showed dose- and time-dependent inhibition of the proliferation of PANC-1 and BxPC3 cells in vitro. Alternol induced apoptosis and cell cycle arrest at S phase and decreased mitochondrial membrane potential. Alternol activated caspase 3, upregulated p53 and p21 expression, and downregulated Bcl-2 expression in a dose-dependent manner. CONCLUSION: Our results suggested that alternol is a candidate for treatment of pancreatic cancer.

Natural compound Alternol induces oxidative stress-dependent apoptotic cell death preferentially in prostate cancer cells.

Prostate cancers at the late stage of castration resistance are not responding well to most of current therapies available in clinic, reflecting a desperate need of novel treatment for this life-threatening disease. In this study, we evaluated the anticancer effect of a recently isolated natural compound, Alternol, in multiple prostate cancer cell lines with the properties of advanced prostate cancers in comparison to prostate-derived nonmalignant cells. As assessed by trypan blue exclusion assay, significant cell death was observed in all prostate cancer cell lines except DU145 but not in nonmalignant (RWPE-1 and BPH1) cells. Further analyses revealed that Alternol-induced cell death was an apoptotic response in a dose- and time-dependent manner, as evidenced by the appearance of apoptosis hallmarks such as caspase-3 processing and PARP cleavage. Interestingly, Alternol-induced cell death was completely abolished by reactive oxygen species scavengers N-acetylcysteine and dihydrolipoic acid. We also demonstrated that the proapoptotic Bax protein was activated after Alternol treatment and was critical for Alternol-induced apoptosis. Animal xenograft experiments in nude mice showed that Alternol treatment largely suppressed tumor growth of PC-3 xenografts but not Bax-null DU-145 xenografts in vivo. These data suggest that Alternol might serve as a novel anticancer agent for patients with late-stage prostate cancer.

Alternol inhibits migration and invasion of human hepatocellular carcinoma cells by targeting epithelial-to-mesenchymal transition.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Such deaths are due, in large part, to its propensity to metastasize. We have examined the effect of alternol on human HCC cells and the underlying molecular mechanism. Therapeutic effects of alternol on cancer cell migration and invasion were analyzed with Boyden chamber and wound healing assays. Effects of alternol on the levels of various proteins involved in cancer cell migration and invasion were determined with gelatin zymography, immunofluorescence, and Western blotting. As shown, treatment with alternol has resulted in a concentration-dependent inhibition of cell migration and invasion of HepG2 cells. The inhibition of HCC invasion by alternol was associated with the suppression of MMP-9 expression and reversal of epithelial-to-mesenchymal transition (EMT). The above results indicated that alternol has the ability to inhibit the migration and invasion of human HCC cells by reversing the process of EMT, suggesting that alternol may be developed as an alternative drug for the treatment of HCC.

Inhibition of human gastric carcinoma cell growth in vitro and in vivo by cladosporol isolated from the paclitaxel-producing strain Alternaria alternata var. monosporus.

Cladosporol was isolated from the fermentation broth of Alternaria alternata var. monosporus obtained from the inner bark of the yew tree and mutated for many generations. We investigated the antitumor effects of cladosporol in vitro and in vivo. The growth-inhibitory effects of cladosporol in vitro against six human cancer cell lines were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The results showed that cladosporol selectively killed cancer cells and had a significant inhibitory effect on the human gastric carcinoma cell line MGC-803 in a concentration- and time-dependent manner. In vivo, cladosporol also showed antitumor activity in nude mice bearing MGC-803 gastric cancer xenografts. These findings suggest that cladosporol has potentially useful growth inhibitory effects on human gastric carcinoma cell lines.

[Apoptosis-inducing effect of alternol on mouse lymphocyte leukemia cells and its mechanism].

Alternol is purified from fermentation productions of microorganisms named as Alternaria alternata var. monosporus. The research is to investigate the apoptosis-inducing effect of alternol on mouse lymphocyte leukemia (L1210) cells and the possible mechanisms. MTT method was used to evaluate the viability of L1210 cells. Apoptosis of L1210 cells was detected by morphological assessment, DNA electrophoresis assay and flow cytometry. Western blotting analysis was carried out to determine the apoptosis-related proteins. Proliferation inhibition of L1210 cells by alternol was found remarkably in a dose-dependent manner. When treated with alternol, apoptotic morphological features of L1210 cells were observed by fluorescent microscopy (AO/EB) and the apoptosis rate was also elevated in a time-dependent manner. After treatments with various concentrations of alternol for 48 h, DNA laddering appeared. The increase of reactive oxygen species (ROS) production was found after cells were exposed to alternol for 6 h, while the decrease of mitochondrial transmembrane potential (delta psi m) was not found until cells were exposed to alternol for 24 h. Furthermore, the level of Bcel-2 and Bcl-2/Bax was down-regulated, while the level of caspase-3 and caspase-9 but not caspase-8 was up-regulated when alternol was added for 72 h. In summary, the results suggested that alternol could inhibit the proliferation of L1210 cells and induce apoptosis of L1210 cells, which was mediated by mitochondria-dependent pathway.