250 S. M; pH 7.4), and 50 g/mL of neutral red containing medium were added. The cells were then subjected to 3 h of incubation. The supernatant was removed, and the cells were washed with a solution of 0.5% CH2O and 1% CaCl2. Subsequently, a solution of 1% CH3COOH and 50% EtOH was added, and the dye was extracted. The absorbance was then read at a wavelength of 550 nm. 3.6. Morphological Analysis Using Phase Contrast Microscopy Changes in morphology were observed to determine the effect of the novel compounds in MCF-7, HepG2 and HEK293 MK-2894 cells. The cells were exposed to different concentrations (10C1000 M) of compounds 3a, 3b and 4 for 24 h. The images were recorded using an inverted phase contrast microscope at 20 magnification. 3.7. Cell Cycle Analysis Measurement of cell cycle arrest was performed using the method of Saquib [37]. Briefly, HepG2 and MCF-7 cells were exposed to numerous concentrations of compounds 3a, 3b and 4 for 24 h. After centrifugation for 4 min at 1000 rpm, cells were fixed with 70% ethanol (500 L) and were then incubated at 4 C for 1 h. The cells were then washed and stained using PBS (500 L; 0.01 M; pH 7.4) containing 50 g/mL propidium iodide (PI), 0.5 mg/mL RNase and Triton X-100. The PI fluorescence was measured using a Beckman Coulter circulation cytometer. The results were MK-2894 analyzed using Coulter Epics XL/XL-MCL, System II Software (Version 3.0, Beckman Coulter, Inc. 250 S. Kraemer Blvd. Brea, CA). 3.8. Apoptosis/Necrosis Assay Using Annexin V-PE and 7-Aminoactinomycin D The assay was performed according to the manufacturers instructions using Annexin V-PE and 7-AAD (Beckman Coulter, Marseille, Cedex 9, France) packages. Briefly, MCF-7 and HepG2 cells MK-2894 were exposed to 250, 500 and 1000 M of compound 3a and 10, 25 or 50 M of 3b and 4 for 24 h. The amount of apoptosis/necrosis in the treated HepG2 and MCF-7 cells was assessed by circulation cytometry using the detailed reported protocol [38]. 3.9. Statistical Analysis ANOVA was utilized for statistical analysis, and results are expressed as the mean standard error of three individual experiments. The treated and control groups were compared using the Dunnetts test. A value of < 0.05 was considered statistically significant. 4. Conclusions This short article explains the synthesis and characterization of novel amidic and acyl urea derivatives of ATRA. The cytotoxicity measurements exhibited a concentration-dependent reduction in the cell viability and alteration of cellular morphology in MCF-7 and HepG2 cells, whereas, in our hands, ATRA was not effective at concentrations ranging MK-2894 from 10C50 M. The use of circulation cytometry to assess Thbs4 cell cycle progression and an apoptosis assay using Annexin V-PE and 7-AAD also revealed that the novel ATRA derivatives possess substantial anticancer activity towards human malignancy cell lines (MCF-7 and HepG2). Arrest in the G2/M phase of the cell cycle could be one of the mechanisms by which cell growth is usually inhibited and apoptosis is usually induced by the compounds. Acknowledgments This research project was supported by a grant from the Research Centre of the Female Scientific and Medical Colleges, Deanship of Scientific Research, King Saud University or college. Supplementary Materials Supplementary can be utilized at: http://www.mdpi.com/1420-3049/20/05/8181/s1. Click here for additional data file.(927K, pdf) Author Contributions Maqsood Ahmed Siddiqui and Nida Nayyar Farshori conceived of and designed the experiments. Maqsood Ahmed Siddiqui, Nida Nayyar Farshori, Quaiser Saquib, Ebtesam Saad Al-Sheddi and Mai Mohammad Al-Oqail performed the experiments. Maqsood Ahmed Siddiqui, Nida Nayyar Farshori, Quaiser Saquib, Ebtesam Saad Al-Sheddi, Mai Mohammad Al-Oqail and Javed Musarrat analyzed the data. Ebtesam Saad Al-Sheddi and Abdulaziz Ali Al-Khedhairy contributed reagents/materials/analysis tools. Maqsood Ahmed Siddiqui and Nida Nayyar Farshori published the paper. All authors read and approved the final manuscript. Conflicts of Interest The authors declare that they have no discord of interest. Footnotes Sample Availability: Samples of the compounds 3aCb and 4 are available from your authors..