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  • Nucleocytoplasmic transport systems play a critical role in


    Nucleocytoplasmic transport systems play a critical role in intracellular localization and regulation of proteins in each cell. This balance is quite important and is typically uncontrolled in cancer cells. Specific localization of tumor suppressor and oncoproteins is quite important because any abnormal localization of these proteins can cause inactivation or over-activation. Hence it was believed that inhibitors of nucleocytoplasmic transport systems could be an anticancer agent for a long time. In this respect, concentration dependent CRM1 inhibitory properties of klavuzons 3, 16b, 16c, 16d and 20 were studied by visualization of CRM1 mediated nuclear export of Riok2 (Serine/threonine-protein kinase Rio2) in HeLa cells. CRM1 inhibitory property of goniothalamin was shown previously and it was used as positive control. DAPI was used to stain the nucleus of the HBTU for comparison purpose. For each molecule, number of cells in three separate pictures were counted and percent inhibition were calculated by dividing the number of CRM1 inhibited cells to the number of total cells. According to the results, goniothalamin started to inhibit CRM1 (50%) at 1.0μM concentration at the end of the 90min of incubation and CRM1 was inhibited completely in 100% of the cell population at 5μM concentration. A longer incubation period (6h) gave similar trend and at 5μM concentration, goniothalamin completely inhibited the nucleocytoplasmic transport system in all cells (Fig. 6). On the other hand, compound 3 started to inhibit CRM1 at 0.2μM concentration even at the end of the 90min of incubation (87%). A similar CRM1 inhibition potency was also found for compounds 16b and 16c. Additionally, compound 16d could not inhibit CRM1 at 0.2μM concentration. However, it inhibited CRM1 completely at 1.0μM concentration at the end of 6h of incubation. Interestingly, compounds 16c and 16d had quite similar IC50 values in MIA PaCa-2 cells (6.30 and 6.70μM, respectively) and it is quite difficult to correlate their CRM1 inhibitory potency with these IC50 values. Lastly compound 20 showed strong CRM1 inhibition activity at 0.2μM concentration at the end of 6h of incubation (Fig. 6). Since compounds 3 and 20 were the most cytotoxic klavuzon derivatives, their CRM1 inhibitory properties were also tested at lower doses (Fig. SM1, Supplementary materials). It was found that both compounds started to inhibit CRM1 at 20nM concentrations, and they almost inhibited CRM1 completely in all cells at 50nM concentrations. Lastly, antiproliferative activity of compound 20 was tested over 3D spheroids generated from HuH-7 hepatocellular cancer cell line in 96 well hanging drop plates in 48h. Then varying concentrations of compound 20 were applied to the spheroids. Goniothalamin was also applied in a single dose just for comparison purpose (Fig. 7). Spheroids were monitored by microscope at the end of 24, 48, and 72h of incubation by using 4X objective, then the same spheroids were treated with propidium iodide (PI) staining and monitored by fluorescence microscope by using 4X objective to visualize the dead cells. As it can be seen in Fig. 7, 50μM concentration of goniothalamin caused significant cell death just after 24h of incubation compared to untreated or DMSO control HuH-7 spheroids. Although its cytotoxic activity did not increase in 48h of incubation, the size of the spheroid was smaller than the sizes of the untreated or DMSO control spheroids. At the end of the 72h of incubation, all of the cells in goniothalamin treated spheroids were dead. On the other hand, compound 20 inhibited the growth of 3D spheroids almost in all tested concentrations and it caused significant cell death at 10 and 30μM concentrations in 48h. Spheroid even started to become disintegrated at 30μM concentration. Similar dose dependent response was observed at the end of 72h of incubation with compound 20.
    Conclusion Synthesis of novel 4′-alkyl substituted klavuzon derivatives were successfully completed in eight steps starting from 4-methyl-1-naphthoic acid. MTT cell viability assay over cancerous (MIA PaCa-2) and immortalized healthy (HPDEC) pancreatic cell lines revealed that compound 20 has a remarkable cytotoxic effect in both cell lines without having any selectivity. Two other novel klavuzon derivatives, 4′-methylklavuzon (3) and 4′-ethylklavuzon (12a), are quite cytotoxic and selective to HPDEC cell line with IC50 values at nanomolar concentrations. Among these, 4′-methylklavuzon (3) and compound 20 were chosen for further biological activity studies and it is found that 4′-methylklavuzon (3) induces apoptosis in MIA PaCa-2 cells at 5 and 10μM concentrations. Comet assay also indicates the formation of single and double strand DNA breaks in MIA PaCa-2 cells treated with 4′-methylklavuzon (3) for 24h. Cell cycle analysis of MIA PaCa-2 cells treated with varying concentrations of compound 3 surprisingly implies the presence of more than one action of mechanism for this compound. At 1μM concentration, compound 3 causes an arrest at G1 phase while at higher concentrations that results in G2 arrest.