Isosteviol derivatives induced apoptosis in Human lung cancer via targeting MEK/MAPK pathway: An in vitro and in vivo study Ahmed M Malki 1,,PhD Stephen C. Bergmeir 2 PhD 1 Department of Health Sciences, Biomedical Program, Faculty of Science, college of Arts and Sciences, Qatar University, Doha, Qatar. 2 Department of Chemistry & Biochemistry, Ohio University, Athens, OH 45701,USA. Background and objectives Experimental Design Conclusion Future directions  The effects of the interesting novel analogs will be screened against various cancer cell lines to investigate their selectivity.  Investigational studies on the mechanism of action of these interesting analogs with more cancer cell lines and in vivo studies are in progress. Results Figure 2 Diterpene derivatives 9 and 15 arrest H1299 cells at G1. The % of cell cycle phases was determined in H1299 treatment with 20 µM and 30 µM of derivatives 9 and 15 respectively for 24 h (A). Each data point was an average of results from thee independent experiments performed in triplicate and presented as M±SD. Cells were treated with derivatives, total protein was extracted and used for Western blotting analysis to determine the differences in p21 expression levels between treated and non-treated cells at the same condition(B), β-actin was used as loading control protein. p21 in H1299 cells was immuno-stained using p21 monoclonal antibody and Texas Red conjugated IgG as the secondary antibody (C). Novel Diterpene derivatives 9 and 15 arrested cells in G1 phase and increased p21 expression Background. Cancer metastasis is the major cause of cancer death. We previously reported novel diterpenes derivatives which induced cytotoxicity in lung cancer cells. The understanding of mechanisms which regulate lung cancer sensitivity to our novel diterpene derivatives is necessary for development of novel set of anticancer derivatives. Aims. Investigate the molecular mechanisms of the optimized ring novel diterpene derivatives on inhibition of proliferation, migration and tumor growth in lung cancer in vitro and in vivo. Results. Our data showed that novel MOM-ether analogs of isosteviol 8c and 9d decreased cell proliferation and induced apoptosis in H1299 lung cancer cells more than p53 stably transfected H1299 cells. Flow cytometric analysis showed that both diterpene derivatives 8c and 9d arrested the H1299 cells in G1 phase which is further confirmed by increased expression level of p21. Moreover, both diterpene derivatives 8c and 9d increased caspase-9 activity in H1299 cells and the induction of apoptosis was significantly reduced after treating cells with caspase-9 inhibitor LEHD-CHO. Both Diterpene derivatives 8c and 9d increased Caspase 3 activities and induced Parp-1 cleavage in H1299 cells. Both derivative 8c and 9d reduced expression levels of AKT and Bcl-2 and increased expression levels of Bax and Bad in H1299cells. Induction of apoptosis was significantly reduced after treating H1299 with AKT inhibitor LY In mice, oral administration of diterpene derivative 9d inhibited the growth of xenograft tumors, invasion, migration, and anchorage-independent growth in tumor tissues without affecting body weight and it decreased the expression levels of VEGF, MMP-9, MEK and MAPK in tumor tissues. Conclusion. Based on previous results, our data support the development of diterpenes derivatives as potential agent for lung cancer treatment via targeting MEK/MAPK pathways As shown in Scheme 2, compound 7 was treated with HCl to remove the MOM group. This provided a small set of analogs containing a free hydroxyl group. The free hydroxyl group was then acylated to provide 12 additional analogs which are screened for anticancer activity. The current study was come out to investigate the effect of novel designed diterpene derivatives on cytotoxicity towards human H1299 lung cancer cells, normal lung epithelial cells (NL-20) and an animal model of lung cancer. The effects of the analogs were investigated by MTT, ELISA-based apoptotic assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, immunofluoresence staining, flow cytometry, real- time reverse transcription polymerase chain reaction and Western blot analysis In vivo studies were performed to assess the anticancer effect of Diterpene induced lung cancer bearing rats and we evaluated its toxicity in different physiological processes. Diterpene derivatives 9 and 15 mediate apoptosis via Akt, caspase-9 and caspase -3 signaling 9d inhibited tumergencity in mice Figure 3 Diterpene derivatives 9 and 15 induced apoptosis via Akt signaling and activation of caspases 9 and 3. H1299 cells treated with Diterpene derivatives 9 and 15 for 24 hr and cells were harvested to investigate expression of Bax, Bid, Akt and pAkt by Western blot analysis (A). Induction of apoptosis was determined after pretreatment of 10 μM LY290042, a representative PI3K/Akt inhibitor for 1 h (B). Induction of apoptosis represents absorbance at 405 nm. Each data point is the mean of three independent experiments and expressed as M±SD. H1299 cells were treated with Diterpene derivatives 9 and 15 and Caspase-9 activity was determined with and without derivatives (C) and H1299 cells were treated with and without Caspase -9 inhibitor and induction of apoptosis was determined (D).Caspase-3 activity (As indicated by (DEVD-pNA) cleavage (E) and Parp-1 cleavage (F) in H1299 cells were determined u[ on treating cells with Diterpene derivatives 9 and 15. Effect of Diterpene analogs on proliferation of lung cancer cells Figure 1 Diterpene derivatives reduced proliferation of H1299 cancer cells. Cell proliferation assay was performed to detect living cells. H1299 cells were treated with 100 µM of the 16 derivatives for 24 h. Cells without derivative treatment were used as control. Each data point was an average of results from thee independent experiments performed in triplicate and presented as M±SD(A). Dose response curve was constructed and the % of viable cells was calculated at each concentration and H1299 ells were treated with various concentrations of the potent derivatives 9 and 15 (B). MTT assay with used to compare the cytotoxicity of Diterpene derivatives 9 and 15 between H1299 cells and NL-20 cells (C). Induction of apoptosis in H1299 cancer cells by the two potent diterpene derivatives according to the chosen concentration for 24 h duration interval. Induction of apoptosis represents absorbance at 405 nm (D). H1299 cells were treated with the same concentrations of derivatives for 24 h and induction of apoptosis was confirmed by appearance of TUNEL positive cells in H1299 cells (E). Figure 4 Effect of Diterpene derivative treatment on growth of tumor volume in control untreated and Diterpene derivative treated group in vivo. The data plotted as mean ±SEM ( n= 10).the tumor volumes of the treated groups were significantly ( P < 0.05 ) lower than the negative control untreated group. Each group of ten rats was used. Cells were treated with derivatives, total protein was extracted and used for Western blotting analysis to determine the differences in VEGF,MEK,ERK, MMP-9 and MAPK expression levels between treated and non-treated cells at the same  Diterpene derivatives 9 and 15 induced apoptosis in H1299 cancer cells more than the normal lung epithelial NL-20 cells. Flow cytometric analysis showed that both Diterpene derivatives 9 and 15 arrested the H1299 cells in G1 phase.  Moreover, both diterpene derivatives increased caspase-9 activity and the induction of apoptosis was significantly reduced after treating cells with caspase-9 inhibitor LEHD-CHO. Both Diterpene derivatives increased Caspase 3 activities and induced Parp-1 cleavage in H1299 cells.  To confirm the involvement of the PI3K/Akt pathway in Diterpene derivatives-induced apoptosis, we investigated whether Derivatives 9 and 15 significantly induces apoptosis in the presence of LY290042, a representative PI3K/Akt inhibitor, co-treatment with LY and Diterpene derivatives resulted in a marked decrease in apoptosis, indicating that the PI3K/Akt pathway plays a role in regulating Diterpene derivatives- induced apoptosis of H1299 cells.  In mice, oral administration of diterpene derivative 9d inhibited the growth of xenograft tumors, invasion and it decreased the expression levels of VEGF, MMP-9, MEK and MAPK in tumor tissues.  Based on previous results, our data support the development of diterpenes derivatives as potential agent for lung cancer treatment via targeting MEK/MAPK pathways Acknowledgements I would like to thank Edison Institute of Biotechnology in Ohio University and Health Sciences Department in Qatar University for supporting this work.