RESUMEN
Here, we present an interesting, previously unreported method for fractionating a particular class of cannabinoids from the crude leaf extract of Cannabis sativa using HP-20 resins. In this study, we report a novel method of divergent synthesis of fractionated Cannabis sativa extract, which allows the generation of multiple cannabinoids C- and O-glycosides which react with the glycosyl donor 2,3,4,6-tetra-O-acetyl-d-mannosyl trichloroacetimidate (TAMTA) to create eight C- and O-ß-d-cannabinoids glycosides (COCG), which are separated by HPLC and whose structures are characterized by 1D, 2D NMR, and mass spectrometry. These glycosides exhibit improved anti-proliferative and anti-metastatic effects against numerous cancer cell lines in vitro and are more water-soluble and stable than their parent cannabinoids. The in vitro testing of the pure cannabinoids (1-4) and their C- & O-glycosides (1a-4a) and 1b-4b exhibited anti-proliferative and anti-metastatic activities against a panel of eight human cancer cell lines in contrast to their respective parent molecules. Different cancer cell lines' IC50 values varied significantly when their cell viability was compared. In addition to the others, compounds 2a, 3a, 4a, and 2b, 3b were highly potent, with IC50values ranging from 0.74 µM (3a) to 51.40 µM (4a).Although2a(1.42 µM) and3a(0.74 µM) exhibited lower IC50values in the MiaPaca-2 cell line than4a(2.58 µM). But, in addition to the comparable anti-clonogenic activity of4ain MiaPaca-2 and Panc-1 cells, it manifested remarkable anti-invasive activity than either 2a or 3a.In contrast to 2a, 2b, 3a, and 3b and their respective parent compounds,4ahad substantial anti-invasive/anti-metastatic capabilities and possessed anti-proliferative activity.The effects of 4a treatment on MiaPaca-2 and Panc-1 cells include a dose-dependent increase in the expression of E-cadherin and a significant decrease in the expression of Zeb-1, Vimentin, and Snail1. Our results demonstrate that divergent synthesis of fractionated Cannabis sativa extract is a feasible and efficient strategy to produce a library of novel cannabinoid glycosides with improved pharmacological properties and potential anticancer benefits.
Asunto(s)
Cannabinoides , Cannabis , Neoplasias , Humanos , Cannabinoides/farmacología , Cannabinoides/química , Cannabinoides/metabolismo , Cannabis/química , Cannabis/metabolismo , Glicósidos/farmacología , Glicósidos/metabolismo , Espectroscopía de Resonancia Magnética , Extractos Vegetales/químicaRESUMEN
Anastasis cascade including induction of Epithelial to Mesenchymal Transition (EMT), DNA repair, and stimulation of pro-survival mediators collectively exaggerate therapy resistance in cancer prognosis. The extensive implications of DNA-damaging agents are clinically proven futile for the rapid development of disease recurrence during treatment regime. Herein we report a glycosidic derivative of Δ9-tetrahydrocannabinol (THC-9-OG) abrogates sub-toxic doses of 5-Fluorouracil (5FU) induced EMT in colon cancer cells nullifying DNA repairing mechanism. Our in vitro and in vivo data strongly proclaims that THC-9-OG could not only abrogate 5FU mediated background EMT activation through stalling matrix degradation as well as murine 4T1 lung metastasis but also vigorously diminished Rad-51 repairing mediator along with stimulation of γ-H2AX foci formation. The combinatorial treatment (5FU + THC-9-OG) in Apc knockout colorectal carcinoma model conferred remission of the crypt progenitor phenotype which was prominently identified in 5FU treatment. Mechanistically, we demonstrated that 5FU plus THC-9-OG significantly attenuated major EMT inducer Vimentin via extensive ROS generation along with autophagy induction via LC3B I-II conversion and p62 degradation in a p-ATM dependent manner. Additionally, Cannabinoid receptor CB1 was responsible for abrogation of Vimentin since we found increase in the expression of γH2AX and decrease in vimentin expression in CB1 agonist (ACEA) plus 5FU treated cells. Nutshell, our results unveil a new direction of Cannabinoid based combinatorial approach to control background EMT along with robust enhancing of DNA damage potential of sub-toxic concentration of 5FU resulting immense inhibition of distant metastasis coupled with triggering cell death in vitro and in vivo.
Asunto(s)
Cannabinoides , Humanos , Animales , Ratones , Fluorouracilo/farmacología , Transición Epitelial-Mesenquimal , Vimentina/genética , Vimentina/metabolismo , Línea Celular Tumoral , Recurrencia Local de Neoplasia , Autofagia , ADNRESUMEN
AKT and ERK 1/2 play a pivotal role in cancer cell survival, proliferation, migration, and angiogenesis. Therefore, AKT and ERK 1/2 are considered crucial targets for cancer intervention. In this study, we envisaged the role of AKT and ERK signaling in apoptosis regulation in presence of compound 4h, a novel synthetic derivative of quinoxalinone substituted spiropyrrolizines exhibiting substantial antiproliferative activity in various cancer cell lines. Structurally 4h is a spiropyrrolizine derivative. Molecular docking analysis revealed that compound 4h shows strong binding affinity with AKT-1 (-9.5 kcal/mol) and ERK2 (-9.0 kcal/mol) via binding at allosteric sites of AKT and active site of ERK2. The implications of 4h binding with these two survival kinases resulted in the obstruction for ATP binding, hence, hampering their phosphorylation dependent activation. We demonstrate that 4h mediated apoptotic induction via disruption in the mitochondrial membrane potential of MCF-7 and HCT-116 cells and 4h-mediated inhibition of survival pathways occurred in a wild type PTEN background and is diminished in PTEN-/- cells. In 4T1 mammary carcinoma model, 4h exhibited pronounced reduction in the tumor size and tumor volume at significantly low doses. Besides, 4h reached the highest plasma concentration of 5.8 µM within a period of 1 h in mice model intraperitoneally. Furthermore, 4h showed acceptable clearance with an adequate elimination half-life and satisfactory pharmacokinetic behaviour, thus proclaiming as a potential lead molecule against breast and colorectal cancer by specifically inhibiting simultaneously AKT and ERK1/2 kinases.
Asunto(s)
Neoplasias de la Mama , Neoplasias Colorrectales , Simulación del Acoplamiento Molecular , Animales , Ratones , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Pirroles/farmacología , Quinoxalinas/farmacología , HumanosRESUMEN
The present work describes the synthesis,conformation and cytotoxic activities of short ß/γ hybrid peptides, Boc-ß2,2-Ac6c-Gpn-NHMe, BG1; Boc-(ß2,2-Ac6c-Gpn)2-OMe, BG2; Boc-(ß2,2-Ac6c-Gpn)3-OMe, BG3; H-ß2,2-Ac6c-Gpn-NHMe, BG4; H-(ß2,2-Ac6c-Gpn)2-OMe, BG5; H-(ß2,2-Ac6c-Gpn)3-OMe, BG6, Boc-ß2,2-Ac6c-Gpn-OMe, BG7 and H-ß2,2-Ac6c-Gpn-OMe, BG8. Mixed C6/C7 conformations were observed for ß/γ hybrid peptides. Further, BG1-BG8 were screened against MCF-7 (Breast cancer), A549 (Lung Cancer), PC-3 (Prostate cancer), HCT-116 (Colon cancer), and MDA-MB-231 (Breast cancer) cell lines. Among all, BG6 exhibited potent cytotoxicity against all cancer cell lines with IC50 ranging from 1.6 µM to 6.3 µM with relatively low cytotoxicity against normal epithelial breast cell line fR-2 and human embryonic kidney cell line HEK-293. Minimal hemolytic activity was observed for BG6 against human erythrocytes. Peptide BG6 displayed anti-migratory and anti-invasive potentials showing strong interactions with intrinsic apoptotic markers Bcl-2, Bax, and cleaved-PARP, as well as the induction of the mitochondria maladjustment mediated apoptosis.
Asunto(s)
Aminoácidos , Neoplasias de la Mama , Humanos , Femenino , Gabapentina , Células HEK293 , Aminoácidos/química , Enlace de Hidrógeno , Cristalografía por Rayos X , Modelos Moleculares , Péptidos/químicaRESUMEN
ATF-4 is a master regulator of transcription of genes essential for cellular-adaptive function. In response to the quantum and duration of stress, ATF-4 diligently responds to both pro-apoptotic and pro-survival signals converging into either autophagy or apoptosis/senescence. Despite emerging cues implying a relationship between autophagy and senescence, how these two processes are controlled remains unknown. Herein, we demonstrate ß-(4-fluorobenzyl) Arteannuin B (here after Arteannuin 09), a novel semisynthetic derivative of Arteannuin B, as a potent ER stress inducer leading to the consistent activation of ATF-4. Persistent ATF-4 expression at early time-points facilitates the autophagy program and consequently by upregulating p21 at later time-points, the signaling is shifted towards G2/M cell cycle arrest. As bZIP transcription factors including ATF-4 are obligate dimers, and because ATF-4 homodimers are not highly stable, we hypothesized that ATF-4 may induce p21 expression by physically interacting with another bZIP family member i.e., C/EBPß. Our co-immunoprecipitation and co-localization studies demonstrated that ATF-4 is principally responsible for the autophagic potential of Arteannuin 09, while as, induction of both ATF-4 and C/EBPß is indispensable for the p21 regulated-cell cycle arrest. Interestingly, inhibition of autophagy signaling switches the fate of Arteannuin 09 treated cells from senescence to apoptosis. Lastly, our data accomplished that Arteannuin 09 is a potent inhibitor of tumor growth and inducer of premature senescence in vivo.