Novel fast dissolving freeze dried sublingual baicalin tablets for enhanced hepatoprotective effect: in-vitro characterization, cell viability, and in-vivo evaluation.

Baicalin (BG), a natural product, has been used in the prevention and treatment of drug-induced liver injury (DILI); however, its poor solubility and extensive liver metabolism limit its pharmacological use. The aim of the present study was the formulation of fast-dissolving freeze-dried sublingual tablets (FFSTs) to increase BG dissolution, avoid first-pass metabolism, and overcome swallowing difficulties. FFSTs were prepared following a 23 factorial design. The effect of three independent variables namely matrix former, Maltodextrin, concentration (4%, and 6%), binder concentration (2%, and 3%), and binder type (Methocel E5, and Methocel E15) on the FFSTs' in-vitro disintegration time and percentage dissolution was studied along with other tablet characteristics. Differential scanning calorimetry, scanning electron microscopy, in-vitro HepG2 cell viability assay, and in-vivo characterization were also performed. F8 (6% Maltodextrin, 2% Mannitol, 2% Methocel E5), with desirability of 0.852, has been furtherly enhanced using 1%PEG (F10). F10 has achieved an in-vitro disintegration time of 41 secs, and 60.83% in-vitro dissolution after 2 min. Cell viability assay, in-vivo study in rats, and histopathological studies confirmed that pretreatment with F10 has achieved a significant hepatoprotective effect against acetaminophen-induced hepatotoxicity. The outcome of this study demonstrated that FFSTs may present a patient-friendly dosage form against DILI.

Thymoquinone Suppresses Angiogenesis in DEN-Induced Hepatocellular Carcinoma by Targeting miR-1-3p.

Hepatocellular carcinoma (HCC) is characterized by its high vascularity and metastasis. Thymoquinone (TQ), the main bio-active constituent of Nigella sativa, has shown anticancer and hepatoprotective effects. TQ's anticancer effect is mediated through miRNA regulation. miR-1-3p plays a significant role in various cancers but its role in HCC invasiveness remains poorly understood. Bio-informatics analysis predicted that the 3'-UTR of TIMP3 is a target for miR-1-3p; Rats were equally divided into four groups: Group 1, the negative control; Group 2 received TQ; Group 3 received DEN; and Group 4 received DEN after pretreatment with TQ. The expression of TIMP3, MMP2, MMP9, and VEGF in rats' liver was determined immunohistochemically. RT-qPCR was used to measure the miR-1-3p level in rats' liver, and TIMP3, MMP2, MMP9, and VEGF in the HepG2 cells after being transfected with miR-1-3p mimic or inhibitor; In rats pretreated with TQ, a decreased expression of MMP2, MMP9 and VEGF, and increased expression levels of TIMP3 and miR-1-3p were detected. Treating the HepG2 cells with miR-1-3p mimic led to the upregulation of TIMP3 and downregulation of MMP2, MMP9, and VEGF, and showed a significant delay in wound healing; These results suggested that the anti-angiogenic effect of TQ in HCC may be mediated through the regulation of miR-1-3p.

Hydroxycitric acid reverses tamoxifen resistance through inhibition of ATP citrate lyase.

Lipid metabolic reprogramming is involved in mediating tamoxifen (TAM) response in breast cancer cells. Published microarray data indicated that ATP citrate lyase (ACLY) is overexpressed in TAM-resistant BC cells. Hydroxycitric acid (HCA) is a powerful competitive inhibitor of the enzyme ACLY, which links carbohydrates and lipids metabolism. However, whether inhibition of ACLY could modulate TAM response in TAM-resistant BC cells remained unexplored. Thus the current study aimed to explore the effect of ACLY inhibition on TAM-resistant BC cells. The cytotoxicity of TAM and/or HCA on LCC2 and its TAM-sensitive counterpart MCF7 cells was evaluated. Also, the effect of TAM and/or HCA treatments on ACLY protein levels were investigated by western blotting. In addition, the effects of TAM and/or HCA on caspase-3, Bax, and Bcl2 levels were evaluated by ELISA.; besides, and flow cytometric analysis was performed for the detection of apoptosis. Moreover, cholesterol and triglyceride contents of LCC2 and MCF7 were quantified colorimetrically. Our results demonstrated that TAM/HCA co-treatment synergistically diminished LCC2 and MCF7 cell viability, with the effect being more significant on LCC2. Mechanistically, TAM/HCA co-treatment decreases the expression level of ACLY in LCC2 by 74 %, while in MCF7 by only 59 %. Moreover, apoptosis marker caspase-3 and Bax were increased, while the anti-apoptotic Bcl2 was decreased. Furthermore, the cholesterol and TG contents were increased in LCC2 than in MCF7. Our data revealed that ACLY plays a key role in TAM resistance and ACLY inhibition by HCA-mediated sensitization of BC-resistant cells to TAM.

Molecular Design, Spectroscopic, DFT, Pharmacological, and Molecular Docking Studies of Novel Ruthenium(III)-Schiff Base Complex: An Inhibitor of Progression in HepG2 Cells.

A novel ruthenium(III)-pyrimidine Schiff base was synthesized and characterized using different analytical and spectroscopic techniques. Molecular geometries of the ligand and ruthenium complex were investigated using the DFT-B3LYP level of theory. The quantum global reactivity descriptors were also calculated. Various biological and molecular docking studies of the complex are reported to explore its potential application as a therapeutic drug. Cytotoxicity of the complex was screened against cancer colorectal (HCT116), breast (MCF-7 and T47D), and hepatocellular (HepG2) cell lines as well as a human normal cell line (HSF). The complex effectively inhibited the tested cancer cells with variable degree with higher activity towards HepG2 (IC50 values were 29 µM for HepG2, 38.5 µM for T47D, 39.7 µM for HCT, and 46.7 µM for MCF-7 cells). The complex induced apoptosis and cell cycle arrest in the S phase of HepG2 cells. The complex significantly induced the expression of H2AX and caspase 3 and caspase 7 gene and the protein level of caspase 3, as well as inhibited VEGF-A and mTOR/AKT, SND1, and NF-kB gene expression. The molecular docking studies supported the increased total apoptosis of treated HepG2 cells due to strong interaction of the complex with DNA. Additionally, the possible binding interaction of the complex with caspase 3 could be responsible for the elevated activity of caspase 3-treated cells. The score values for the two receptors were -3.25 and -3.91 kcal/mol.

In Vitro Cytotoxic Activity and Phytochemical Characterization (UPLC/T-TOF-MS/MS) of the Watermelon (Citrullus lanatus) Rind Extract.

Reusing food waste is becoming popular in pharmaceutical industries. Watermelon (Citrullus lanatus) rind is commonly discarded as a major solid waste. Here, the in vitro cytotoxic potential of watermelon rind extracts was screened against a panel of human cancer cell lines. Cell cycle analysis was used to determine the induction of cell death, whereas annexin V-FITC binding, caspase-3, BAX, and BCL-2 mRNA expression levels were used to determine the degree of apoptosis. VEGF-promoting angiogenesis and cell migration were also evaluated. Moreover, the identification of phytoconstituents in the rind extract was achieved using UPLC/T-TOF-MS/MS, and a total of 45 bioactive compounds were detected, including phenolic acids, flavonoids aglycones, and their glycoside derivatives. The tested watermelon rind extracts suppressed cell proliferation in seven cancer cell lines in a concentration-dependent manner. The cytotoxicity of the rind aqueous extract (RAE) was higher compared with that of the other extracts. In addition to a substantial inhibitory effect on cell migration, the RAE triggered apoptosis in HCT116 and Hep2 cells by driving the accumulation of cells in the S phase and elevating the activity of caspase-3 and the BAX/BCL-2 ratio. Thus, a complete phytochemical and cytotoxic investigation of the Citrullus lanatus rind extract may identify its potential potency as an anticancer agent.

Targeting CDK7 reverses tamoxifen resistance through regulating stemness in ER+ breast cancer.

BACKGROUND: Although tamoxifen is the mainstay endocrine therapy for estrogen receptor-positive (ER+) breast cancer patients, the emergence of tamoxifen resistance is still the major challenge that results in treatment failure. Tamoxifen is very effective in halting breast cancer cell proliferation; nonetheless, the ability of tamoxifen to target cancer stem and progenitor cell populations (CSCs), a major key player for the emergence of tamoxifen resistance, has not been adequately investigated yet. Thus, we explored whether targeting CDK7 modulates CSCs subpopulation and tamoxifen resistance in ER+ breast cancer cells. METHODS: Mammosphere-formation assay, stem cell biomarkers and tamoxifen sensitivity were analyzed in MCF7 tamoxifen-sensitive cell line and its resistant counterpart, LCC2, following CDK7 targeting by THZ1 or siRNA. RESULTS: Analysis of clinically relevant data indicated that expression of stemness factor, SOX2, was positively correlated with CDK7 expression in tamoxifen-treated patients. Moreover, overexpression of the stemness gene, SOX2, was associated with shorter overall survival in those patients. Importantly, the number of CSC populations and the expression of CDK7, P-Ser118-ER-α and c-MYC were significantly higher in LCC2 cells compared with parental MCF-7 cells. Moreover, targeting CDK7 inhibited mammosphere formation, CSC-regulating genes, and CSC biomarkers expression in MCF-7 and LCC2 cells. CONCLUSION: Our data indicate, for the first time, that CDK7-targeted therapy in ER+ breast cancer ameliorates tamoxifen resistance, at least in part, by inhibiting cancer stemness. Thus, targeting CDK7 might represent a potential approach for relieving tamoxifen resistance in ER+ breast cancer.

Conversion of RNA Aptamer into Modified DNA Aptamers Provides for Prolonged Stability and Enhanced Antitumor Activity.

Aptamers, synthetic single-strand oligonucleotides that are similar in function to antibodies, are promising as therapeutics because of their minimal side effects. However, the stability and bioavailability of the aptamers pose a challenge. We developed aptamers converted from RNA aptamer to modified DNA aptamers that target phospho-AXL with improved stability and bioavailability. On the basis of the comparative analysis of a library of 17 converted modified DNA aptamers, we selected aptamer candidates, GLB-G25 and GLB-A04, that exhibited the highest bioavailability, stability, and robust antitumor effect in in vitro experiments. Backbone modifications such as thiophosphate or dithiophosphate and a covalent modification of the 5'-end of the aptamer with polyethylene glycol optimized the pharmacokinetic properties, improved the stability of the aptamers in vivo by reducing nuclease hydrolysis and renal clearance, and achieved high and sustained inhibition of AXL at a very low dose. Treatment with these modified aptamers in ovarian cancer orthotopic mouse models significantly reduced tumor growth and the number of metastases. This effective silencing of the phospho-AXL target thus demonstrated that aptamer specificity and bioavailability can be improved by the chemical modification of existing aptamers for phospho-AXL. These results lay the foundation for the translation of these aptamer candidates and companion biomarkers to the clinic.

Blockade of CDK7 Reverses Endocrine Therapy Resistance in Breast Cancer.

Cyclin-dependent kinase (CDK)-7 inhibitors are emerging as promising drugs for the treatment of different types of cancer that show chemotherapy resistance. Evaluation of the effects of CDK7 inhibitor, THZ1, alone and combined with tamoxifen is of paramount importance. Thus, in the current work, we assessed the effects of THZ1 and/or tamoxifen in two estrogen receptor-positive (ER+) breast cancer cell lines (MCF7) and its tamoxifen resistant counterpart (LCC2) in vitro and in xenograft mouse models of breast cancer. Furthermore, we evaluated the expression of CDK7 in clinical samples from breast cancer patients. Cell viability, apoptosis, and genes involved in cell cycle regulation and tamoxifen resistance were determined. Tumor volume and weight, proliferation marker (Ki67), angiogenic marker (CD31), and apoptotic markers were assayed. Bioinformatic data indicated CDK7 expression was associated with negative prognosis, enhanced pro-oncogenic pathways, and decreased response to tamoxifen. Treatment with THZ1 enhanced tamoxifen-induced cytotoxicity, while it inhibited genes involved in tumor progression in MCF-7 and LCC2 cells. In vivo, THZ1 boosted the effect of tamoxifen on tumor weight and tumor volume, reduced Ki67 and CD31 expression, and increased apoptotic cell death. Our findings identify CDK7 as a possible therapeutic target for breast cancer whether it is sensitive or resistant to tamoxifen therapy.

Part III: Novel checkpoint kinase 2 (Chk2) inhibitors; design, synthesis and biological evaluation of pyrimidine-benzimidazole conjugates.

Recently a dramatic development of the cancer drug discovery has been shown in the field of targeted cancer therapy. Checkpoint kinase 2 (Chk2) inhibitors offer a promising approach to enhance the effectiveness of cancer chemotherapy. Accordingly, in this study many pyrimidine-benzimidazole conjugates were designed and twelve feasible derivatives were selected to be synthesized to investigate their activity against Chk2 and subjected to study their antitumor activity alone and in combination with the genotoxic anticancer drugs cisplatin and doxorubicin on breast carcinoma, (ER+) cell line (MCF-7). The results indicated that the studied compounds inhibited Chk2 activity with high potency (IC50 = 5.56 nM - 46.20 nM). The studied candidates exhibited remarkable antitumor activity against MCF-7 (IG50 = 6.6  µM - 24.9 µM). Compounds 10a-c, 14 and 15 significantly potentiated the activity of the studied genotoxic drugs, whereas, compounds 9b and 20-23 antagonized their activity. Moreover, the combination of compound 10b with cisplatin revealed the best apoptotic effect as well as combination of compound 10b with doxorubicin led to complete arrest of the cell cycle at S phase where more than 40% of cells are in the S phase with no cells at G2/M. Structure-activity relationship was discussed on the basis of molecular modeling study using Molecular modeling Environment program (MOE).

Anticancer potentiality of lignan rich fraction of six Flaxseed cultivars.

The objective of our study is to highlight the therapeutic effect and mechanism of action by which purified Flaxseed hydrolysate (PFH) which is a lignan rich fraction exerts its anticancer activity on a human breast cancer cell line (T47D) and in mice bearing tumor. HPLC analysis of PFH of six flaxseed cultivars had shown that PFH of the cultivar Giza 9 (PFH-G9) contains the highest concentration of SDG (81.64 mg/g). The in vitro cytotoxic potentiality of PFH's of six flaxseed cultivars was screened against a panel of human cancer cell lines. PFH -G9 showed the most significant cytotoxic activity against ER-receptor positive breast cell lines MCF7 and T47D with IC50 13.8 and 15.8 µg/ml, respectively. Moreover, PFH-G9 reduced the expression of the metastasis marker, 1-α, metalloproteinases and vascular endothelial growth factor (VEGF), one of the most potent stimulators of angiogenesis, while it increased the caspase-3 dependent apoptosis. Our study also showed that dietary intake of 10% of Giza 9 Flaxseeds (FS), fixed oil (FSO) or Flax meal (FSM) twice daily for 3 weeks in mice-bearing solid Ehrlich ascites carcinoma (EAC) resulted in reducing the tumor volume, the expression of estrogen, insulin growth factor, progesterone, VEGF and MMP-2, but enhanced expression of caspase-3.

Part II: New candidates of pyrazole-benzimidazole conjugates as checkpoint kinase 2 (Chk2) inhibitors.

The development of checkpoint kinase 2 (Chk2) inhibitors for the treatment of cancer has been an ongoing and attractive objective in drug discovery. In this study, twenty-one feasible pyrazole-benzimidazole conjugates were synthesized to study their effect against Chk2 activity using Checkpoint Kinase Assay. The antitumor activity of these compounds was investigated using SRB assay. A potentiation effect of the synthesized Chk2 inhibitors was also investigated using the genotoxic anticancer drugs cisplatin and doxorubicin on breast carcinoma, (ER+) cell line (MCF-7). In vivo Chk2 and antitumor activities of 8d as a single-agent, and in combination with doxorubicin, were evaluated in breast cancer bearing animals induced by N-methylnitrosourea. The effect of 8d alone and in combination with doxorubicin was also studied on cell-cycle phases of MCF-7 cells using flow cytometry analysis. The results revealed their potencies as Chk2 inhibitors with IC50 ranges from 9.95 to 65.07 nM. Generally the effect of cisplatin or doxorubicin was potentiated by the effect of most of the compounds that were studied. The in vivo results indicated that the combination of 8d and doxorubicin inhibited checkpoint kinase activity more than either doxorubicin or 8d alone. There was a positive correlation between checkpoint kinase inhibition and the improvement observed in histopathological features. Single dose treatment with doxorubicin or 8d produced S phase cell cycle arrest whereas their combination created cell cycle arrest at G2/M from 8% in case of doxorubicin to 51% in combination. Gold molecular modelling studies displayed a high correlation to the biological results.

Part I: Design, synthesis and biological evaluation of novel pyrazole-benzimidazole conjugates as checkpoint kinase 2 (Chk2) inhibitors with studying their activities alone and in combination with genotoxic drugs.

Activated checkpoint kinase 2 (Chk2) is a tumor suppressor as one of the main enzymes that affect the cell cycle. 2-Biarylbenzimidazoles are potent selective class of Chk2 inhibitors; the structure-based design was applied to synthesize a new series of this class with replacing the lateral aryl group by substituted pyrazoles. Ten pyrazole-benzimidazole conjugates from the best fifty candidates according to docking programs have been subjected to chemical synthesis in this study. The activities of the conjugates 5-14 as checkpoint kinase inhibitors and as antitumor alone and in combination with genotoxic drugs were evaluated. The effect of compounds 7 and 12 on cell-cycle phases was analyzed by flow cytometry analysis. Antitumor activity of compounds 7 and 12 as single-agents and in combinations with doxorubicin was assessed in breast cancer bearing animals induced by MNU. The Results indicated that compounds 5-14 inhibited Chk2 activity with high potency (IC50 52.8 nM-5.5 nM). The cytotoxicity of both cisplatin and doxorubicin were significantly potentiated by the most of the conjugates against MCF-7 cell lines. Compounds 7 and 12 and their combinations with doxorubicin induced the cell cycle arrest in MCF-7 cells. Moreover, compound 7 exhibited marked higher antitumor activity as a single agent in animals than it's combination with doxorubicin or doxorubicin alone. The combination of compound 12 with doxorubicin was greatly effective on animal than their single-dose treatment. In conclusion, pyrazole-benzimidazole conjugates are highly active Chk2 inhibitors that have anticancer activity and potentiate activity of genotoxic anticancer therapies and deserve further evaluations.

Anticancer Activities of New N-hetaryl-2-cyanoacetamide Derivatives Incorporating 4,5,6,7-Tetrahydrobenzo[b]thiophene Moiety.

AIMS: Novel series of N-(4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl) cyanoacetamide derivatives are synthesized. METHOD: The structure of these compounds was elucidated using different spectral tools. Compounds were evaluated for their cytotoxic activities against different types of human cancer cell lines including, breast (MCF-7, T47D, MDA MB231); liver (HEPG-2); colon (HCT116); prostate (PC3); and cervix (HELA) cells. In this study, we used compounds 11 and 12 that showed the highest cytotoxicity on PC3 and HEPG2 cells, to explore their effects on apoptosis, metastasis and angiogenesis of cancer cells. RESULTS: Results revealed that the growth inhibition produced by the two selected compounds was due to cytocidal and not due to cytostatic effect in both cell lines. This cytocidal effect was due to up-regulation of caspases-3, and- 9. In addition, the two compounds inhibited the expression of metalloproteinases-2 and 9 (MMP 2&9). Moreover, HIF-1alpha and VEGF expressions were inhibited by both compounds. CONCLUSION: In conclusion, N-(4, 5, 6, 7-tetrahydrobenzo[b]thiophen-2-yl) cyanoacetamide derivatives showed different anticancer potential against different cancer cell lines. Compounds 11 and 12 showed the most active cytotoxicity against PC3 and HepG2 cells. Both compounds have apoptotic, anti- metastatic and anti-angiogenic effects.

Targeting glycolysis by 3-bromopyruvate improves tamoxifen cytotoxicity of breast cancer cell lines.

BACKGROUND: Tamoxifen is the standard endocrine therapy for ER+ breast cancer; however, many women still relapse after long-term therapy. 3-Bromopyruvate, a glycolytic inhibitor, has shown high selective anti-tumor activity in vitro, and in vivo. The aim of this study was to evaluate the possible augmentation of the effect of tamoxifen via reprograming cancer cell metabolism using 3-bromopyruvate. METHODS: An in vitro screening of antitumor activity as well as the apoptotic, anti-metastatic, and anti-angiogenic potentials of the combination therapy were carried out using different techniques on breast cancer cell lines MCF7and T47D. In addition the antitumor effect of the combined therapy was done on mice bearing tumor. RESULTS: Our results showed modulation in apoptosis, angiogenesis and metastatic potential by either drug alone; however, their combination has surpassed that of the individual one. Combination regimen enhanced activated caspases-3, 7 and 9, as well as oxidative stress, signified by increased malondialdehyde and decreased glutathione level. Additionally, the angiogenesis and metastasis markers, including hypoxia inducing factor-1α, vascular endothelia growth factor, and metaloproteinases-2 and 9 were decreased after using the combination regimen. These results were further confirmed by the in vivo study, which depicted a decrease in the tumor volume and angiogenesis and an increase in oxidative stress as well. CONCLUSION: 3-bromopyruvate could be a valuable compound when added with tamoxifen in breast cancer treatment.

Cytotoxic and antimicrobial evaluations of novel apoptotic and anti-angiogenic spiro cyclic 2-oxindole derivatives of 2-amino-tetrahydroquinolin-5-one.

A novel series of cyclic 2-oxindole derivatives incorporating 2-amino-tetrahydroquinolin-5-one were prepared. The structures of the prepared compounds were elucidated using different spectral tools. The regio-orientation of the reaction products was elucidated through NOE difference experiments and through using substituents on the ortho position to affect further cyclization. Antitumor and antimicrobial evaluations were performed on the prepared compounds. Most of these compounds exhibited high to moderate antimicrobial activity. With respect to the antitumor activity, the compounds showed more potent cytotoxic effect only toward the human breast cancer cell line MCF-7. Also, we found that derivatives containing an ester group (8c, 11b, 14b, and 15b) are more active than those containing a cyanide group (8a, 11a, 14a, and 15a). Moreover, compounds 15b and 8b are the most active derivatives in this group. These two compounds showed apoptotic inhibition of the proliferation of human breast adenocarcinoma MCF-7 cells through DNA fragmentation, induction of the tumor suppressor protein p53, induction of caspase-9, and finally the inhibition of angiogenesis by decreasing vascular endothelial growth factor expression and secretion.