The multi-parametric analysis provided quantitative information on the changes in five commonly used cellular parameters , including cell viability, membrane permeability, mitochondria membrane permeability, cytochrome c, and nuclear intensity. See Figures 1-4 for the quantitative results.
This is a little studied bush between 1.2 to 3 meters high, its leaves are composed with 5 to 10 leaflets, which in the dorsal part are green and in the underside are brown-yellowish, with blooms between February and April 4 . In Hidalgo, it is located in the municipalities of Ixmiquilpan, San Salvador and Tepetitlan 5 . Due to the popular use of this species as an antitumor and the little information about it, in the present work the antineoplastic effect was observed in a murine lymphoma model, as well as its secondary metabolites and acute toxicity in aqueous and ethanolic extracts, since a wider knowledge about this resource could lead to a safer and more effective use by the population.
The tumor tissues obtained from the mice that received treatment with NS, Blank NP, Sal free 2, Sal NP 2, and Sal NP 8 were selected for further study. These tissues were ground into cell suspensions. Cells were washed twice with phosphate-buffered saline (PBS) solution and lysed with RIPA Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai, China) and protease inhibitor (Thermo Fisher Scienti ﬁ c, USA). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scienti ﬁ c, USA). Equivalent amounts of total protein (60 µg) were boiled and electrophoretically separated on a 10% polyacrylamide gel at 80 volts. The proteins were trans- ferred onto polyvinylidene di ﬂ uoride membranes. The membranes were blocked for 60 min with a 5% milk solu- tion prepared in PBS, incubated overnight at 4°C with primary antibodies (GAPDH, CD44, CD133, E-cadherin, VIM, ZEB1, and ZEB2) at a 1:500 dilution. The membranes were washed thrice for 5 min each time with Tween 20 (1:1000 dilution)-PBS and incubated for 45 min with the appropriate peroxidase-conjugated secondary antibody (Abcam, USA) (1:1000 dilution). Membranes were washed with Tween 20-PBS thrice for 10 min each time and visua- lized using the Odyssey two-color infrared laser imaging system (ECDOI, Greenville, USA). The signal generated by GAPDH was used as an internal control.
To explore whether the antitumoreffect of artesunate on gastric cancer cells is mediated through its inhibitory effect on COX-2 expression, the viability of HGC-27 cells was assessed by MTT assay after treatment with various concen- trations of celecoxib (0, 20, 40, and 80 μ mol/L), a well-known inhibitor of COX-2, from 24 to 72 hours. Treatment of the cells with celecoxib resulted in a dose- and time-dependent reduction in the cell viability of HGC-27 cells as compared with non-celecoxib-treated controls (P,0.05) (Figure 4). Celecoxib also could induce cell apoptosis (Figure 5). Treatment of the HGC-27 cells with celecoxib for 48 hours induced a marked, dose-dependent induction of both the early and late stages of apoptosis. Celecoxib treatment increased the number of apoptotic cells from 8.83% in the untreated cell group to 25.86% in the group treated with 80 μ mol/L celecoxib. These data suggest that the inhibition of COX-2 expression is linked to the inhibition of cell proliferation and induction of cell apoptosis.
night at 4°C. Membranes were then incubated with primary antibody using anti- β -actin, anti-p53, anti-p21, anti-Bax, anti-bcl2, anti-caspase-3 and anti-caspase-9 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) at a concentration of 1:500. After washing the membranes for 1 h using 1 × PBS with 0.5% Tween 20, they were incubated with secondary antibody (obtained from Promega, Madison, WI, USA) at a concentration of 1:2500 for 1 h at room temperature. The membranes were then washed and the development was done using Western blotting chemiluminescent reagent enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA) He and figures were taken using ChemiDoc XRS + machine (Bio- Rad, Hercules, CA, USA).
To evaluate the effect of inhibitors on the cellular uptake of iRGD-modified SSLs, B16-F10 cells were preincubated with different inhibitors for 30 minutes at 37 ° C. Briefly, the cells were incubated for 24 hours, then chlorpromaz- ine (10 µ g/mL), amiloride (20 µ M), filipin (5 µ g/mL), or M- β -CD (2.5 mM) was added, followed by incubation for 30 minutes at 37 ° C. After that, the inhibitor-containing cul- ture medium was replaced with iRGD-SSL-coumarin-6 (the final concentration of coumarin-6 was 150 ng/mL) and incubated for 2 hours at 37 ° C. Then, the cells were washed three times with PBS solution, harvested by trypsinization, and centrifuged at 1,000 rpm for 5 minutes. After being resuspended in 500 µ L PBS medium, the cells were tested using a FACScan™ (BD Biosciences, San Jose, CA, USA). The coumarin-6 in the cells was excited with an argon laser (467 nm), and fluorescence was detected at 502 nm. To investigate the effect of temperature on cellular uptake, the cells without inhibitor treatment were incubated at both 37 ° C and 4 ° C, and treated as described earlier.
Objective: Breast cancer has been reported to be a serious disease and a threat to women’s health. 2,3,5,4′-Tetrahydroxystilbene-2-O-b-D-glucoside (THSG) is a bioactive natural compound originating from Polygonum multiflorum Thunb., which has been shown to possess anti-inflammatory and antitumor properties. Adriamycin (ADM) is a chemotherapy agent used in tumor therapy that is limited by its side effects. However, little is known about the syner- gistic effect of THSG combined with ADM on breast cancer. This study seeks to investigate the effects of the combination of THSG plus ADM on MCF-7 breast cancer cells and to test the mechanisms involved.
This difference in cellular localization between ATF– HSA:DOX and DOX also helps to answer a question involv- ing FCS. Ten percent FCS was included in all of the in vitro experiments to facilitate cell culture. FCS contains bovine serum albumin (BSA). Will BSA affect the in vitro experi- ments by binding to DOX in the control experiment or even extract DOX out of ATF–HSA:DOX complex? We think this effect is small. If DOX would be in complex with BSA and enter cells, it would be quite likely to localize in cytoplasm, similar to ATF–HSA:DOX, which was not the case observed. This result here provides support that DOX is likely not in complex with BSA in the settings of our in vitro experi- ments. On another hand, BSA binding to DOX is weaker than HSA (the binding constant K DOX-HSA = 1.1 × 10 4 M -1 ,
As seen in Figure 7, the most effective therapeutics was the DOX/TPL-1/0.2-LPNPs-treated group, with an obvious inhibition of tumor growth during the whole treatment. The tumor volume of DOX/TPL-1/0.2-LPNPs-treated group was only 34.0% of control group at the end of experiment, which was 5.5-, 4.5-, and 2.0-fold smaller than that treated with free DOX, free TPL, and free DOX/TPL, respectively. A combination of synergistic effects, passive targeting, and responsive release would mainly explain the highest antitu- mor effect of DOX/TPL-1/0.2-LPNPs. It is noteworthy that DOX/TPL-1/0.2-LPNPs possessed higher antitumor capac- ity than the combination of DOX-LPNPs and TPL-LPNPs. The results might be attributed to the higher synergistic efficiency of codelivery system. With the assistance of the codelivery system, DOX and TPL were transported to the tumor site and released in the same cancer cell. Compared to the mixture of single drug-loaded system, the codelivery system can preferably play the synergistic effect of the drug combination, which was consistent with expected.
EMT is an important cellular process associated with poor prognosis in HCC patients and mediates metastasis. 48–53 During this process, cell adhesion-related proteins are down- regulated, whereas mesenchymal-related proteins are upregu- lated. These effects result in the loss of cell polarity and insensitivity to antitumor drugs. 54,55 It has been shown that EMT is a key factor contributing to the development of resis- tance to sorafenib. 56 – 59 Therefore, disruption of the EMT process is a promising strategy to enhance the sensitivity of HCC cells to molecular targeted agents. In the present study, we observed that treatment with chelidonine restricted EMT by modulating its key regulators. Moreover, chelidonine func- tioned as an antitumor agent through the following mechan- isms: (1) Reduction of telomere length; (2) inhibition of the tumor necrosis factor- α /nuclear factor- κ B pathways; (3) induc- tion of mitotic slippage and apoptotic-like death; or (4) inhibi- tion of the formation of the integrin-linked kinase/PINCH/ α -
The computer aided prediction of biological activity in relation to the chemical structure of a compound is now commonly used technique in drug discovery. Moreover, understanding the QSAR of known compounds helps to facilitate the new drug discovery. Computational chemistry represents molecular structures as a numerical model and simulates their behavior with the equations of quantum and classical physics. The available programs enable scientists to easily generate and present molecular data including geometries, energies and associated properties (electronic, spectroscopic and bulk). The usual paradigm for displaying and manipulating these data is a table in which compounds are defined by individual rows and the molecular properties (or descriptors) are defined by the associated columns [8-10].
Performance of animal experiments was reviewed and approved by the Institutional Animal Care and Use Committee of the Beijing Tiantan Hospital. All animal experi- ments were performed in accordance with the UK Animals (Scienti ﬁ c Procedures) Act, 1986, and its associated guide- lines. Nude mice aged 4 – 6 weeks were purchased from Si- Bei-Fu Biotechnology Corporation, Beijing China. For the subcutaneous tumor model, 27,28 cells were transfected with miRNA-940 or miR-940 + mutated MACC1 (MACC1 Mut ) and injected into nude mice (1×10 6 cells per animal). After 2 – 4 days, animals received oral administration of 1 mg/kg dose Anlotinib per two days. After 3 – 4 weeks ’ growth, the mice were harvested, and tumor weights were measured. Tumor volumes were calculated following the methods provided by Wang et al (2018) and Chen et al (2018). 27,28
BALB/c nude mice were divided into two groups in parallel for survival detection and pathological experimentation. The mice in the survival group were not killed, they were allowed to die naturally, and those in the pathological group were killed. Survival and death of the mice bearing lung tumor were recorded throughout the administration course. At the end of the treatment, the mice were killed for weighing the lung tissues with tumor. Furthermore, we used hematoxylin and eosin (HE) staining to observe the pathological changes of lung tissues and employed immunohistochemical (IHC) technique to detect the expressions of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), Bcl-2, and P53 in tumor tissue for confirming the antitumoreffect of BJO-CN in the orthotopic lung cancer model. Random images (400 × ) per experiment group were captured using a microscope equipped with the analysis software Image- Pro-Plus (MediaCybernetics, Rockville, MD, USA). The semi quantification of protein expression was determined by analyzing the mean integrated optical density (IOD) in a randomly selected manner.
Briefly, cells were harvested by scraping and centrifugation (700 ×g, 2 minutes) and washed with PBS. Cell suspensions were mixed with liquefied Comet Agarose at a 1:10 ratio (v/v) and pipetted on an OxiSelect Comet Slide (75 µ L/well). After a 15-minute embedding step (4 ° C, dark, horizontal posi- tion), cells were lysed (25 mL lysis buffer/slide, 30-minute incubation, 4 ° C, dark, horizontal position) and treated with an alkaline solution (25 mL/slide, 30 minutes, 4 ° C, dark) to relax and denature the DNA. Finally, the samples are electrophoresed in a horizontal chamber (300 mA for 30 minutes) to separate intact DNA from damaged fragments.
Allium sativum L. or garlic (family: Liliaceae) has been used as a flavoring agent, food and for its medicinal properties in many diseases all over the world (1-4). The Allium genus include more than 600 various species and garlic is a member of this genus and other members are leek, chive, shallot and scallion. They are different in appearance, color and flavor but similar in biochemical and phytochemical constituent (4, 5). Garlic has been applied for the treatment of several illnesses since ancient times (6). In folk remedy garlic is applied for treatment of many diseases such as respiratory illnesses, asthma, diabetes, cardiovascular diseases, rheumatism, infections, cancers and gastrointestinal disorder (spleen and liver diseases), inflammations, leucoderma, and also it is utilize as a tonic, aphrodisiac, emmenogogue and anthelmentic (1, 7- 9). Recent pharmacological studies have demonstrated insecticidal (10), antihypoxic (11), antibacterial (12), antifungal (13), hypoglycemic (14), hypolipidemic (15), and antiatherosclerotic (3) and antihepatotoxic (16) activities of garlic.
The PDX tumor models mostly resemble their original tumor, which is in line with our western blot and immunohistochemistry staining (Figure S1D-G). The activities of CDK1, PDK1 and β-Catenin are specific similar between the clinical tissues and paired PDX models (Figure S1E). To explore the direct effect of CDK1 inhibitor alone or combined with sorafenib, we applied them to F1 generation PDX tumor models in vivo (Figure 2A). The results showed that tumor growth was suppressed by 75%, 49% and 92% in RO3306, sorafenib, and combinatorial treatment groups. Particularly, in the RO3306 combined with sorafenib group, a synergistic anticancer effect on the overall PDX tumor models was revealed (Figure 2B-C). The HCC case #4 PDX model illustrated a dramatically synergistic reduction in tumor growth in line with the overall PDX tumors. HCC case #10 PDX model displayed tumor resistance to sorafenib treatment, whereas combinatorial treatment showed a significant enhanced antitumoreffect, overcoming the tumor resistance (Figure 2D-E). Both PDX case #4 and case #10 were consistent with their tumor volume growth curves (Figure S2A-B). Taken together, the CDK1-targeted inhibitor can enhance the efficacy of sorafenib treatment, which would be applied to overcome its limited effect and improve the outcome of advanced HCC patients. In addition, the HCC case #9 PDX model indicated a dramatic resistance to RO3306, sorafenib, and combinatorial treatment, which we presumed is due to low tumor progression and growth (Figure S2C-D). Slow PDX tumor growth was also observed in aggressive triple negative breast cancer (HCI-004 of supplementary Figure 2 in that paper) , which makes it different than effective suppression. Moreover, with under treatment alone or in combination, the mouse body weights of various treatment groups had no significant difference, indicating the drug tolerance at the beginning of administration (Figure S2E).
Isopeptides ( ε -peptides) of lysine, with a given Mw and low polydispersity (10–400 units), were synthesized to study the relationship between their chemical structure and biological effect. The designed compounds were of high purity, low polydispersity and high stereochemical purity. The effect of the compounds was tested on a human erythroleukemia cell line (K-562) and on four transplantable mouse tumors (L1210 lymphoid leukemia, P38 macrophage derived tumor, Ehrlich ascites carcinoma, Lewis lung tumor /LLT/). In case of the L1210 and P388 tumors and the Ehrlich carcinoma, survival of the animals was used as an indicator of the effect. In case of the Lewis lung tumor, the number and size of metastases in the lung and/or liver of treated and untreated mice were used as indicators. The polymers of polymerisation degree 80–120 (Mw 10.2–15.4 KD) showed the strongest antiproliferative effect both on K562 cells and the tumors growing in vivo. This effect was manifest with a significantly higher survival rate as compared to the control (L1210, P38, Ehrlich ascites), furthermore, by a decrease in the number and size of liver and lung metastases (LLT).
Figure 6. ICOS transmembrane domain is necessary for improved antitumoreffect and increased persistence. (A) Surface expression of the CAR proteins on human T cells at the time of functional evaluation. ICOSBBz has an ICOS transmembrane region and TM CD8–ICOSBBz and BBICOSz have a CD8α trans- membrane domain. Transduction efficiencies are indicated with MFI of the transduced populations in parentheses. (B) Fold-change CAR expression (MFI) in CD8-ICOSBBz and BBICOSz relative to ICOSBBz was analyzed in 3 normal donors. *P < 0.05 by 1-way ANOVA with Tukey post hoc test. (C) T cell volume during ex vivo expansion in the absence of cognate antigen. Representative of 2–4 donors (D) Representative histograms showing the expression of activa- tion, differentiation, and exhaustion markers in CAR T cells 13 days after stimulation with anti-CD3/CD28 beads. Representative of 2 donors. (E) CAR T cells were cocultured with APC cells transduced with mesothelin (K562meso). Supernatants were obtained 24 hours later, and cytokine production was analyzed by Luminex. (F and G) NSG mice bearing s.c. Capan-2 pancreatic tumors were treated 36 days after tumor implantation with 2 doses of UTD or CAR T cells. (F) Tumor volume was analyzed at indicated time points. Results are expressed as a mean tumor volume (± SEM) with n = 5–7 mice per group. *P < 0.05, **P < 0.01 by 2-way repeated-measure ANOVA. (G) The concentration of CD4 + T cells and CD8 + T cells was determined in the blood of treated animals 21 days
cells were harvested and washed in 1 × phosphate-buffered saline (PBS). Total RNA was extracted from infected cells by TRIZOL (Invitrogen). Then, 1 μ g of total RNA and the oligo (dt) primer was applied for reverse-transcription polymerase chain reaction (RT-PCR) assay utilizing Two Step RT-PCR kit (TakaRa, Otsu, Japan), and the procedure was done according to the manufacturer’s instructions. Dm- dNK gene was amplified by PCR in the following process: 94 ° C for 4 minutes, 35 cycles at 94 ° C for 1 minute each, 60 ° C for 1 minute, and 1.5 minute at 72 ° C. The GAPDH primers were: sense, 5 ′ -ACC ACA GTC CAT GCC ATC AC-3 ′ ; and antisense, 5 ′ -TCC ACC ACC CTG TTG CTG TA-3 ′ . The Dm-dNK primers were: sense, 5 ′ CCG GAA TTC ACC ATG GCG GAG GCA 3 ′ ; and antisense, 5 ′ CGC GGA TCC TCA TTA TCT GGC GAC 3 ′ . Finally, 1.5% agarose gel electrophoresis was done for visualing the amplification products.
The continuous demand for new chemotherapeutic agents indicates that new approaches are critically needed. The current study was under taken to examine the effect of the two novel antitumor complexes namely cobalt and chromium complexes of bis-(4-bromobenzaldehydeiminoacetophenone), BBIA-Co and BBIA-Cr, on liver, kidney and heart of Ehrlich ascites carcinoma (EAC) bearing male albino mice. The effect of the two complexes on antioxidant status of the animals and histopathological examination of liver, kidney and heart tissues were also examined. Results indicated that treatment with either BBIA-Co or BBIA-Cr had ameliorated to some extent the changes in liver function exerted by EAC inoculation. Both complexes had no significant effect on kidney function, while they induced cardiac toxicity to animals. The study also showed no significant changes in the antioxidant status of EAC inoculated mice treated with BBIA-Co compared to normal animals, which was not indicated after BBIA-Cr treatment. The biochemical data results were further supported by histopathological examination.