Senolytic Cocktail Dasatinib+Quercetin (D+Q) Does Not Enhance the Efficacy of Senescence-Inducing Chemotherapy in Liver Cancer

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death, which develops in the context of fibrosis and cirrhosis caused by chronic inflammation, in turn due to non-alcoholic fatty liver disease (NAFLD), alcohol consumption and/or hepatitis viral infection. An increased number of senescent cells are associated with age-related tissue degeneration during NAFLD-induced HCC, or during chemotherapeutic treatment. Senolytic agents target selectively senescent cells. A combination of the senolytic drugs dasatinib and quercetin (D+Q) reduced hepatic lipid accumulation and alleviated age-associated physical dysfunction in mice. However, whether D+Q can impact the treatment of HCC, at the end-stage of the NAFLD inflammatory spectrum, is unknown. Here, using two well-established HCC cell lines (HepG2, Huh-7), we demonstrate that the maximal cytostatic doses for D and/or Q (1 + 1 μM) lacked efficacy in removing doxorubicin-induced β-gal-positive senescent cells. Moreover, D+Q did not affect doxorubicin-dependent induction of flattened morphology, activation of p16, expression of SASP-associated genes or formation of γH2AX foci. We then investigated the antitumor efficacy of doxorubicin, D+Q, or the combination, in xenograft studies conducted with HCC cells inoculated in athymic nude mice. Doxorubicin reduced tumor growth by 30% compared to control mice, while D+Q was ineffective in synergizing with doxorubicin and in clearing doxorubicin-induced HCC senescent cells. Unexpectedly, D+Q alone appeared to have acute pro-tumorigenic effects in control mice. While our data need to be confirmed in animal models that fully recapitulate NAFLD, we demonstrate that these compounds are ineffective, alone or in synergy with senescence-inducing chemotherapy, against experimental HCC.

Isolation of senescent cells by iodixanol (OptiPrep) density gradient‐based separation

“…Interestingly, the appearance and enrichment of DXR‐induced senescence markers were more prominent in HepG2 cells compared with Huh‐7 cells. Huh‐7 cells are more aggressive than HepG2 cells, in vitro and in vivo, and the two cell lines have a different genetic make‐up . While HepG2 cells express wild‐type p53, Huh‐7 cells express a dominant‐negative mutant form of p53 (Y220C), which may be involved in resistance to DXR‐induced p53‐mediated cell senescence …”

“…Huh-7 cells are more aggressive than HepG2 cells, in vitro and in vivo, and the two cell lines have a different genetic make-up. 9,19,36,37 While HepG2 cells express wild-type p53, Huh-7 cells express a dominant-negative mutant form of p53 (Y220C), 38 which may be involved in resistance to DXR-induced p53-mediated cell senescence. 39 An increased number of senescent cells are associated not only to chemotherapeutic treatment but also to age-related tissue degeneration towards HCC.…”

“…This concentration is only slightly cytotoxic to HCC cells, inducing senescence in ~40% of cells. 9,15,19 We then washed out the DXR and incubated the cells in normal medium for a further 5 days. Upon trypsin-mediated cell detachment,…”

“…The DXR-induced senescent cells and control (untreated) cells were processed for staining using the fluorogenic substrate of SA-β-galactosidase (C12FDG, or FDG) and for flow cytometry analysis, as we previously described. 9 The following cell mixtures were prepared: (a) 60:40 (60% of control cells and 40% of DXR-induced cells); (b) 50:50 (50% of control cells and 50% of DXR-induced cells); (c) 30:70 (30% of control cells and 70% of DXR-induced cells). The samples were divided into two parts: one part was fixed immediately (UNS) and the other part was processed thought gradient centrifugation (SEP).…”

“…7 These features are of interest because they can be exploited for selective killing or clearing of senescent cells using a heterogeneous class of drugs known as senolytics. 8,9 Attempts to isolate senescent cells using surface markers (DcR2, DPP4, oxidized vimentin) [10][11][12] or fluorescent ubiquitination-based cell-cycle indicator technology 13 have shown promise. However, simple methods to isolate and culture senescent cells-despite their low or null proliferative capacity-are lacking, particularly in the context of chemotherapy-induced senescence for cancer treatment.…”