Please cite this article as: S. Moccia, M. Russo, M. Durante, et al., A carotenoid-enriched extract from pumpkin delays cell proliferation in a human chronic lymphocytic leukemia cell line through the modulation of autophagic flux, Current Research in Biotechnology (2020), https://doi.Journal Pre-proof J o u r n a l P r e -p r o o f 2 Abstract Chronic lymphocytic leukemia (CLL) is the most frequent form of leukemia in adult population. From an asymptomatic state, which can lay for years, CLL

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... idly evolves becoming fatal. Apoptosis resistance and induction of a protective form of autophagy are possible explanations of the poor responsivity of CLL to conventional and innovative therapeutic drugs. Carotenoids, including and -carotene, lycopene, and derivatives, such as retinoic acid, have been studied for their significant antiproliferative and differentiating activity in cancer. Here, we report that a carotenoid-enriched extract (CE) obtained from pumpkin by supercritical CO 2 extraction showed an anti-proliferative effect on HG3 cell line derived from human B-CLL cells. CE induced a 40% delay in cell proliferation compared to untreated cells, without signs of cytotoxicity. This delay was associated with p27 Kip1 over-expression, AMPK activation and modulation of autophagy flux. In HG3 cells treated with CE, we detected a 30% autophagosome intracellular increase and changes in the expression of canonical biochemical markers of autophagy (LC3-II, p62). These results suggest the presence in the CE of a pool of bioactive carotenoids acting additively or synergistically in retarding cancer cell growth. Journal Pre-proof J o u r n a l P r e -p r o o f 2020; Hou et al., 2013; Russo et al., 2017c; Shen et al., 2014). We previously reported that a carotenoid enriched extract (CE), obtained by supercritical CO 2 (SC-CO 2 ) from Cucurbita moschata sp., exerted an anti-proliferative effect on different malignant cell line through the Journal Pre-proof J o u r n a l P r e -p r o o f 6 activation of a "non-protective" form of autophagy (Russo et al., 2017). To our knowledge, no studies investigated the effects of a mixture of carotenoids in CLL-derived cell lines or in ex vivo models of B-cells isolated from patients. To verify the capacity of CE to interfere with cell growth in CLL-derived cell lines, we employed in the present study HG3 cells established from a CLL clone by EBV infection (Rosen et al., 2012). Journal Pre-proof J o u r n a l P r e -p r o o f