Total Flavonoids in Artemisia absinthium L. and Evaluation of Its Anticancer Activity.

To overcome the shortcomings of traditional extraction methods, such as long extraction time and low efficiency, and considering the low content and high complexity of total flavonoids in Artemisia absinthium L., in this experiment, we adopted ultrasound-assisted enzymatic hydrolysis to improve the yield of total flavonoids, and combined this with molecular docking and network pharmacology to predict its core constituent targets, so as to evaluate its antitumor activity. The content of total flavonoids in Artemisia absinthium L. reached 3.80 ± 0.13%, and the main components included Astragalin, Cynaroside, Ononin, Rutin, Kaempferol-3-O-rutinoside, Diosmetin, Isorhamnetin, and Luteolin. Cynaroside and Astragalin exert their cervical cancer inhibitory functions by regulating several signaling proteins (e.g., EGFR, STAT3, CCND1, IGFIR, ESR1). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the anticancer activity of both compounds was associated with the ErbB signaling pathway and FoxO signaling pathway. MTT results showed that total flavonoids of Artemisia absinthium L. and its active components (Cynaroside and Astragalin) significantly inhibited the growth of HeLa cells in a concentration-dependent manner with IC50 of 396.0 ± 54.2 µg/mL and 449.0 ± 54.8 µg/mL, respectively. Furthermore, its active components can mediate apoptosis by inducing the accumulation of ROS.

Cecropin-Loaded Zeolitic Imidazolate Framework Nanoparticles with High Biocompatibility and Cervical Cancer Cell Toxicity.

Cecropins (CECs) are insect venom-derived amphiphilic peptides with numerous pharmacological effects, including anti-inflammatory, antibacterial, antiviral, and anti-tumor activities. Cecropins induce tumor cell death by disrupting phospholipid membrane integrity. However, non-specific cytotoxicity and in vivo rapid degradation limit clinical application. Nanotechnologies provide novel strategies for tumor eradication, including nanocarriers that can precisely target drugs to tumor tissue. We report the fabrication of CEC-encapsulated zeolitic imidazolate framework 8 (ZIF-8) nanoparticles (CEC@ZIF-8 NPs) via the preparation of CEC@ZIF-8 NPs in pure water by one-pot stirring. This method yielded morphologically uniform NPs with 20 wt% drug loading capacity and 9% loading efficiency. The NP formulation protected CECs from proteasome degradation, enhanced peptide bioavailability, promoted HeLa tumor cell uptake, and increased antitumor efficacy compared to free CECs. In conclusion, this ZIF-8 encapsulation strategy may enhance the clinical applicability of CECs and other antitumor peptides.

The Extracts of Artemisia absinthium L. Suppress the Growth of Hepatocellular Carcinoma Cells through Induction of Apoptosis via Endoplasmic Reticulum Stress and Mitochondrial-Dependent Pathway.

Artemisia absinthium L. has pharmaceutical and medicinal effects such as antimicrobial, antiparasitic, hepatoprotective, and antioxidant activities. Here, we prepared A. absinthium ethanol extract (AAEE) and its subfractions including petroleum ether (AAEE-Pe) and ethyl acetate (AAEE-Ea) and investigated their antitumor effect on human hepatoma BEL-7404 cells and mouse hepatoma H22 cells. The cell viability of hepatoma cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis, cell cycle, mitochondrial membrane potential (Δψm), and reactive oxygen species (ROS) were analyzed by flow cytometry. The levels of proteins in the cell cycle and apoptotic pathways were detected by Western blot. AAEE, AAEE-Pe, and AAEE-Ea exhibited potent cytotoxicity for both BEL-7404 cells and H22 cells through the induction of cell apoptosis and cell cycle arrest. Moreover, AAEE, AAEE-Pe, and AAEE-Ea significantly reduced Δψm, increased the release of cytochrome c, and promoted the cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP) in BEL-7404 and H22 cells. AAEE, AAEE-Pe, and AAEE-Ea significantly upregulated the levels of ROS and C/EBP-homologous protein (CHOP). Further, AAEE, AAEE-Pe, and AAEE-Ea significantly inhibited tumor growth in the H22 tumor mouse model and improved the survival of tumor mice without side effects. These results suggest that AAEE, AAEE-Pe, and AAEE-Ea inhibited the growth of hepatoma cells through induction of apoptosis, which might be mediated by the endoplasmic reticulum stress and mitochondrial-dependent pathway.

Cistanche tubulosa phenylethanoid glycosides induce apoptosis in H22 hepatocellular carcinoma cells through both extrinsic and intrinsic signaling pathways.

BACKGROUND: Cistanche tubulosa (Schenk) R. Wight is a traditional Chinese medicine that parasitizes the roots of the Tamarix plant and has been used to treat male impotence, sterility, body weakness, and as a tonic. However, its antitumor effect on hepatocellular carcinoma is still elusive. Here, we investigated the antitumor effect of C. tubulosa phenylethanoid glycosides (CTPG) on H22 hepatocellular carcinoma cells both in vitro and in vivo and its mechanisms. METHODS: The morphology, viability, apoptosis, cell cycle and mitochondrial membrane potential (Δψm) of H22 cells were analyzed by inverted microscopy, MTT assay and flow cytometry, respectively. The expression and activation of proteins in apoptosis pathway were detected by Western blot. The in vivo antitumor effect was evaluated in tumor mouse model established using male Kunming mice. RESULTS: CTPG treatment significantly suppressed H22 cell growth in a dose- and time-dependent manner, which was correlated with the increased apoptosis and cell cycle arrest at G0/G1 and G2/M phases. Moreover, the chromosomal condensation was observed in CTPG-treated H22 cells. CTPG treatment significantly increased Bax/Bcl-2 ratio, reduced Δψm and enhanced the release of cytochrome c. The levels of cleaved caspase-8 and caspase-9 in both extrinsic and intrinsic signaling pathways were significantly increased that sequentially activated caspase-7 and -3 to cleave PARP. Finally, CTPG inhibited the growth of H22 cells in mice and improved the survival rate of tumor mice. CONCLUSIONS: These results suggested that CTPG suppressed H22 cell growth through both extrinsic and intrinsic apoptosis pathways.

CecropinXJ, a silkworm antimicrobial peptide, induces cytoskeleton disruption in esophageal carcinoma cells.

Antimicrobial peptides exist in the non-specific immune system of organism and participate in the innate host defense of each species. CecropinXJ, a cationic antimicrobial peptide, possesses potent anticancer activity and acts preferentially on cancer cells instead of normal cells, but the mechanism of cancer cell death induced by cecropinXJ remains largely unknown. This study was performed to investigate the cytoskeleton-disrupting effects of cecropinXJ on human esophageal carcinoma cell line Eca109 using scanning electron microscopy observation, fluorescence imaging, cell migration and invasion assays, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. The electronic microscope and fluorescence imaging observation suggested that cecropinXJ could result in morphological changes and induce damage to microtubules and actin of Eca109 cells in a dose-dependent manner. The cell migration and invasion assays demonstrated that cecropinXJ could inhibit migration and invasion of tumor cells. Western blot and qRT-PCR analysis showed that there was obvious correlation between microtubule depolymerization and actin polymerization induced by cecropinXJ. Moreover, cecropinXJ might also cause decreased expression of α-actin, ß-actin, γ-actin, α-tubulin, and ß-tubulin genes in concentration- and time-dependent manners. In summary, this study indicates that cecropinXJ triggers cytotoxicity in Eca109 cells through inducing the cytoskeleton destruction and regulating the expression of cytoskeleton proteins. This cecropinXJ-mediated cytoskeleton-destruction effect is instrumental in our understanding of the detailed action of antimicrobial peptides in human cancer cells and cecropinXJ might be a potential therapeutic agent for the treatment of cancer in the future.

Non-viral vectors combined delivery of siRNA and anti-cancer drugs to reverse tumor multidrug resistance.

Multidrug resistance (MDR) of tumors is one of the main reasons for the failure of chemotherapy. Multidrug resistance refers to the cross-resistance of tumor cells to multiple antitumor drugs with different structures and mechanisms of action. Current strategies to reverse multidrug resistance in tumors include MDR inhibitors and RNAi technology. siRNA is a small molecule RNA that is widely used in RNAi technology and has the characteristics of being prepared in large quantities and chemically modified. However, siRNA is susceptible to degradation in vivo. The effect of siRNA therapy alone is not ideal, so siRNA and anticancer drugs are administered in combination to reverse the MDR of tumors. Non-viral vectors are now commonly used to deliver siRNA and anticancer drugs to tumor sites. This article will review the progress of siRNA and chemotherapeutic drug delivery systems and their mechanisms for reversing multidrug resistance.

Research Progress of Plant-Derived Natural Products against Drug-Resistant Cancer.

As one of the malignant diseases globally, cancer seriously endangers human physical and mental health because of its high morbidity and mortality. Conventional cancer treatment strategies, such as surgical resection and chemoradiotherapy, are effective at the early stage of cancer but have limited efficacy for advanced cancer. Along with cancer progress and treatment, resistance develops gradually within the population of tumor cells. As a consequence, drug resistance become the major cause that leads to disease progression and poor clinical prognosis in some patients. The mechanisms of cancer drug resistance are quite complex and involve various molecular and cellular mechanisms. Therefore, exploring the mechanisms and finding specific targets are becoming imperative to overcome drug resistance. In recent years, plant-derived natural products have been evaluated as potential therapeutic candidates against cancer with drug resistance due to low side effects and high anticancer efficacy. A growing number of studies have shown that natural products can achieve superior antitumor effects through multiple signaling pathways. The mechanisms include regulation of multiple drug resistance (MDR)-related genes, inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, induction of autophagy, and blockade of the cell cycle. This paper reviews the molecular and cellular mechanisms of cancer drug resistance, as well as the therapeutic effects and mechanisms of plant-derived natural products against cancer drug resistance. It provides references for developing therapeutic medication for drug-resistant cancer treatment with high efficacy and low side effects.

Phytosphingosine inhibits the growth of lung adenocarcinoma cells by inducing G2/M-phase arrest, apoptosis, and mitochondria-dependent pathway cell death in vitro and in vivo.

In order to search for novel antitumor drugs with high efficiency and low toxicity, the anti-lung cancer activity of phytosphingosine was studied. Phytosphingosine is widely distributed in fungi, plants, animals, and has several biological activities, including anti-inflammation and anti-tumor. However, its anti-lung cancer activity needs to be further investigated. The effects and pharmacological mechanisms of phytosphingosine on lung cancer treatment were investigated both in vitro and in vivo. The results showed that phytosphingosine inhibited the growth of lung cancer cell lines. Phytosphingosine induced apoptosis through a mitochondria-mediated pathway, phytosphingosine arrested the cell cycle at the G2/M phase and induced apoptosis in a dose-dependent manner by increasing Bax/Bcl-2 ratio, which caused the decrease of mitochondrial membrane potential to promote the release of cytochrome C, caspase 9 and 3, and degrade PARP in A549 cells. The results showed that phytosphingosine could damage the mitochondrial functions, increase ROS levels, and arrest the cell cycle at the G2/M stages. Finally, phytosphingosine also inhibited the growth of tumor in mice. Taken together, phytosphingosine suppressed the growth of lung cancer cells both in vitro and in vivo and had potential application in the research and development of antitumor drugs. The aim of the present study was to explain the theoretical basis of phytosphingosine therapy for lung cancer and providing new possibilities for lung cancer treatment.

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity.

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.

Expression, purification and characterization of cecropin antibacterial peptide from Bombyx mori in Saccharomyces cerevisiae.

CecropinXJ is a cationic antimicrobial peptide originally isolated from the larvae of Bombyx mori. In this study, an antibacterial peptide gene of cecropinXJ was cloned into the pYES2/CT/α Factor expression vector and expressed in the Saccharomyces cerevisiae INVSc1 strain. Following an induction of recombinant protein expression in yeast for 120 h, the maximum amount of total secreted protein was 1.437 g/L. The percentage of recombinant cecropinXJ was estimated to be 79.45% of the total protein. After purification with Ni-NTA agarose column, recombinant cecropinXJ was noted to exert strong antimicrobial activities against a broad-spectrum of microorganisms, including Gram-negative and Gram-positive bacteria. Its minimal inhibitory concentration (MIC) against Escherichia coli ATCC25922 was 0.81 µM. In addition, transmission electron microscopy (TEM) analysis indicated that the surfaces of the treated pathogens underwent obvious morphological changes compared with the untreated controls, suggesting that this antimicrobial peptide exerts its action by directly disrupting membranes of microorganisms. CecropinXJ had a small hemolytic effect on red blood cells even with a peptide concentration of 200 µM. Thus, cecropinXJ acts selectively on bacterial membranes. Purified recombinant antibacterial peptide, cecropinXJ, retained a high stability against E. coli ATCC25922 over a temperature range from 4 °C to 100 °C and a pH range from pH 2.0 to 12.0. Taken together, this study demonstrates that recombinant cecropinXJ can be produced in large quantities in yeast with genetic engineering methods, and that it has strong and rapid antimicrobial activities against all of microorganisms tested. Our results suggest that cecropinXJ is a potential candidate for therapy.

Plant-derived natural products and combination therapy in liver cancer.

Liver cancer is one of the malignant cancers globally and seriously endangers human health because of its high morbidity and mortality. Plant-derived natural products have been evaluated as potential anticancer drugs due to low side effects and high anti-tumor efficacy. However, plant-derived natural products also have defects of poor solubility and cumbersome extraction process. In recent years, a growing numbers of plant derived natural products have been used in combination therapy of liver cancer with conventional chemotherapeutic agents, which has improved clinical efficacy through multiple mechanisms, including inhibition of tumor growth, induction of apoptosis, suppression of angiogenesis, enhancement of immunity, reversal of multiple drug resistance and reduction of side effects. The therapeutic effects and mechanisms of plant-derived natural products and combination therapy on liver cancer are reviewed to provide references for developing anti-liver-cancer strategies with high efficacy and low side effects.

Potential Mechanisms of Plant-Derived Natural Products in the Treatment of Cervical Cancer.

Cervical cancer is the second most common gynecological malignancy globally; it seriously endangers women's health because of its high morbidity and mortality. Conventional treatments are prone to drug resistance, recurrence and metastasis. Therefore, there is an urgent need to develop new drugs with high efficacy and low side effects to prevent and treat cervical cancer. In recent years, plant-derived natural products have been evaluated as potential anticancer drugs that preferentially kill tumor cells without severe adverse effects. A growing number of studies have shown that natural products can achieve practical anti-cervical-cancer effects through multiple mechanisms, including inhibition of tumor-cell proliferation, induction of apoptosis, suppression of angiogenesis and telomerase activity, enhancement of immunity and reversal of multidrug resistance. This paper reviews the therapeutic effects and mechanisms of plant-derived natural products on cervical cancer and provides references for developing anti-cervical-cancer drugs with high efficacy and low side effects.

The petroleum ether extract of Brassica rapa L. induces apoptosis of lung adenocarcinoma cells via the mitochondria-dependent pathway.

Brassica rapa L. is one of the most popular traditional foods with a variety of biological activities. In this study, the petroleum ether extract of B. rapa was separated by silica gel column chromatography, and named BRPS, which was identified by LC-MS. The effects and pharmacological mechanisms of BRPS on the treatment of lung cancer were investigated both in vitro and in vivo. The results showed that BRPS significantly inhibited the proliferation of both human lung cancer A549 and mouse lung cancer LLC cells, while its toxicity to normal cells was lower than that of cancer cells. BRPS induced cell cycle arrest at the G2/M phase and significantly reduced the levels of CDK1 and CyclinB1 in A549 cells. Moreover, BRPS induced apoptosis in a dose-dependent manner, and increased the Bax/Bcl-2 ratio, while it decreased mitochondrial membrane potential, promoted the release of cytochrome c, activated caspase 9 and 3, and enhanced the degradation of PARP in A549 cells. Furthermore, the levels of reactive oxygen species (ROS) were also upregulated by BRPS and ROS inhibitor reversed BRPS-induced apoptosis. Importantly, BRPS significantly suppressed the growth of LLC cells in vivo without any obvious side effect on body weight and organs of mice, and increased the proportion of B cells, CD4+ T cells, CD8+ T cells and CD44+CD8+ T cells in the spleen. These results revealed that BRPS inhibited the growth of lung cancer cells through inducing cell cycle arrest, mitochondria-dependent apoptosis, and activating immunity of mice, and BRPS might be a potential anti-tumor functional food and promising agent for the treatment of lung cancer.

Cistanche tubulosa Phenylethanoid Glycosides Induce Apoptosis of Hepatocellular Carcinoma Cells by Mitochondria-Dependent and MAPK Pathways and Enhance Antitumor Effect through Combination with Cisplatin.

Cistanche tubulosa is a type of Chinese herbal medicine and exerts various biological functions. Previous studies have been demonstrated that Cistanche tubulosa phenylethanoid glycosides (CTPG) exhibit antitumor effects on a variety of tumor cells. However, the antitumor effects of CTPG on HepG2 and BEL-7404 hepatocellular carcinoma (HCC) cells are still elusive. Our study showed that CTPG significantly inhibited the growth of HepG2 and BEL-7404 cells through the induction of cell cycle arrest and apoptosis, which was associated with the activation of MAPK pathways characterized by the up-regulated phosphorylation of p38, JNK, and ERK1/2 and mitochondria-dependent pathway characterized by the reduction of mitochondrial membrane potential. The release of cytochrome c and the cleavage of caspase-3, -7, -9, and PARP were subsequently increased by CTPG treatment. Moreover, CTPG significantly suppressed the migration of HepG2 through reducing the levels of matrix metalloproteinase-2 and vascular endothelial growth factor. Interestingly, CTPG not only enhanced the proliferation of splenocytes but also reduced the apoptosis of splenocytes induced by cisplatin. In H22 tumor mouse model, CTPG combined with cisplatin further inhibited the growth of H22 cells and reduced the side effects of cisplatin. Taken together, CTPG inhibited the growth of HCC through direct antitumor effect and indirect immunoenhancement effect, and improved the antitumor efficacy of cisplatin.

Marchantia polymorpha L. ethanol extract induces apoptosis in hepatocellular carcinoma cells via intrinsic- and endoplasmic reticulum stress-associated pathways.

BACKGROUND: Marchantia polymorpha L. is a kind of Chinese herbal medicine and has various biological activities including antioxidant and antifungal. However, it is not clear about the antitumor effect and mechanism of M. polymorpha. We prepared M. polymorpha ethanol extract (MPEE) and investigated its antitumor effect on hepatocellular carcinoma cells both in vitro and in vivo. METHODS: The viability of hepatocellular carcinoma cells was detected by MTT assay. The distribution of cell cycle was analyzed by propidium iodide (PI) staining. The morphology of nuclei was observed by Hoechst 33258 staining. Apoptosis was detected by Annexin V/PI staining. JC-1 fluorescent probe and DCFH-DA were used to detect the mitochondrial membrane potential (ΔψM) and the level of reactive oxygen species (ROS), respectively. Caspase inhibitors were used to test the function of caspase in the induction of apoptosis. Quantitative real time polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the levels of mRNA and protein, respectively. Differentially expressed genes and signaling pathways were identified by transcriptome analysis. The H22 tumor mouse model was used to detect the antitumor effect of the extract. RESULTS: MPEE significantly suppressed the migration and growth of BEL-7404, HepG2 and H22 cells in a dose- and time-dependent manner through induction of apoptosis characterized by chromosomal condensation and cell cycle arrest at G0/G1 and G2/M phases. MPEE induced mitochondria-dependent apoptosis via upregulation of Bax and downregulation of Bcl-2 to reduce mitochondrial membrane potential and increase the release of cytochrome c. The levels of cleaved caspase-8 and -9 were significantly increased, which sequentially activated caspase-3 to cleave PARP. We further found that MPEE significantly increased ROS production and activated endoplasmic reticulum (ER) stress associated-apoptotic signaling pathway. Moreover, MPEE significantly inhibited H22 tumor growth in mouse model and improved the survival of tumor mice. CONCLUSION: These results suggested that MPEE suppressed hepatocellular carcinoma cell growth through induction of apoptosis via intrinsic- and ER stress-associated pathways.

Prevalence and Factors Associated With Suicidal Ideation in Medical Students With Migraine.

Background: The association between migraine and suicide ideation has been identified. However, the predictive factors of suicidal ideation are still controversial and whether migraine with aura can serve as an independent associated factor is uncertain. This manuscript studied the association between migraine with aura and suicidal ideation and explored the predictive factors for suicidal ideation. Methods: We surveyed 9,057 medical students and included 579 medical students with migraine into our study population. All students completed the General Situation Questionnaire, the Verified Headache Questionnaire, Hamilton Anxiety Scale (24 items), Hamilton Depression Scale (24 items), 36-item Health Survey Brief (SF-36), Headache Impact Text-6 (HIT-6), Test Anxiety Scale (TAS), and Pittsburgh Sleep Quality Index (PSQI). Suicidal ideation was measured by the Self-rating Idea of Suicide Scale (SIOSS). Results: Out of the 579 migraine medical college students, 562 (age 19.6 ± 1.6; 448 women and 114 men) were included in the final study. The positive rate of suicidal ideation was 13.7%. Compared with students suffering from migraine without aura, those having migraine with aura had higher suicidal ideation (p < 0.015). After adjusting for demographic factors and headache characteristics, migraine with aura was found to be independently associated with suicidal ideation. Other independent associated factors include anxiety, depression, test anxiety, sleep, headache, and quality of life. Among these various factors, high quality of life was found to play a protective role against suicidal ideation. Conclusions: Migraine with aura is independently associated with suicidal ideation. Furthermore, anxiety, depression, text anxiety, poor sleep quality, and headache frequency are associated with suicidal ideation among medical college students with migraine.

Influence of magnolol on the secretion of alpha-toxin by Staphylococcus aureus.

In this study we investigated the antimicrobial activity of magnolol on Staphylococcus aureus. The minimal inhibitory concentrations of magnolol against 31 S. aureus strains ranged from 4-32 microg/mL. In addition, hemolysin assays, Western blotting, and real-time RT-PCR were performed to investigate the effect of magnolol on alpha-toxin secretion by both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). The results indicated that sub-inhibitory concentrations of magnolol dose-dependently inhibited the transcription of hla (the gene encoding alpha-toxin) in S. aureus, resulting in a reduction of alpha-toxin secretion and, thus, hemolytic activities.

Preparation and Characterization of Anti-GPC3 Nanobody Against Hepatocellular Carcinoma.

BACKGROUND: Glypican-3 (GPC3) is a newly identified target molecule for the early diagnosis of hepatocellular carcinoma (HCC), while targeted inhibition of GPC3 signaling may help to control the proliferation and metastasis of HCC cells. The purpose of this study was to prepare the anti-GPC3 nanobody and to investigate the affinity of the anti-GPC3 nanobodies in vitro and the anticancer effects on hepatocellular carcinoma in vivo. METHODS: To screen for unknown anti-GPC3 antibodies, we constructed an antibody phage display library. After three rounds of panning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA). Further, the nanobody fusion protein was expressed in E. coli BL21 cells and purified by affinity chromatography. Competitive ELISA and flow cytometry were conducted to confirm the affinity of the anti-GPC3 nanobodies in vitro. The antitumor effects of VHHGPC3 were assessed in vivo. RESULTS: The results showed that the nanobody VHHGPC3 had specific high-affinity binding to His-GPC3 antigen. Moreover, VHHGPC3 exhibited specific binding to commercial human GPC3 and recognized the surface GPC3 protein of the hepatoma cell line HepG2. Importantly, in vivo study showed that GPC3 nanobody suppresses the growth of HepG2 and improves the survival rate of tumor mice. DISCUSSION: In summary, our new anti-GPC3 nanobody suggests a strong application potential for targeted therapy of liver cancer.

Cistanche tubulosa phenylethanoid glycosides induce apoptosis in Eca-109 cells via the mitochondria-dependent pathway.

Cistanche tubulosa has various biological functions. In the present study, the antitumor effect of water-soluble phenylethanoid glycosides of C. tubulosa (CTPG-W) on esophageal cancer was investigated. Eca-109 cells were treated with CTPG-W and the cell viability was measured by MTT assay. The apoptosis, cell cycle, mitochondrial membrane potential (Δψm) and reactive oxygen species were analyzed by flow cytometry. The levels of proteins in apoptotic pathways were detected by western blot analysis. It was determined that CTPG-W significantly reduced the viability of Eca-109 cells through the induction of apoptosis and cell cycle arrest. Following CTPG-W treatment, the Δψm of Eca-109 was notably decreased, which is associated with the upregulated levels of B-cell lymphoma-2 (Bcl-2)-associated X and downregulated levels of Bcl-2. Consequently, the levels of cytochrome c and c-Jun NH2-terminal kinase were increased, which upregulated the levels of cleaved-poly (ADP-ribose) polymerase and cleaved-caspase-3, -7 and -9, but not caspase-8. Correspondingly, the levels of reactive oxygen species in Eca-109 cells demonstrated notable changes. These results indicated that CTPG-W induced apoptosis of Eca-109 cells through a mitochondrial-dependent pathway.