Introduction: Venetoclax (VCX) is a selective BCL-2 inhibitor approved for the treatment of leukemia and lymphoma. However, the mechanisms of anti-cancer effect of VCX either as a monotherapy or in combination with other chemotherapeutic agents against breast cancer need investigation. Methods: Breast cancer cell lines with different molecular subtypes (MDA-MB-231, MCF-7, and SKBR-3) were treated with different concentrations of VCX for indicated time points. The expression of cell proliferative, apoptotic, and autophagy genes was determined by qRT-PCR and Western blot analyses. In addition, the percentage of MDA-MB-231 cells underwent apoptosis, expressed higher oxidative stress levels, and the changes in the cell cycle phases were determined by flow cytometry. Results: Treatment of human breast cancer cells with increasing concentrations of VCX caused a significant decrease in cells growth and proliferation. This effect was associated with a significant increase in the percentage of apoptotic MDA-MB-231 cells and in the expression of the apoptotic genes, caspase 3, caspase 7, and BAX, with inhibition of antiapoptotic gene, BCL-2 levels. Induction of apoptosis by VCX treatment induced cell cycle arrest at G0/G1 phase with inhibition of cell proliferator genes, cyclin D1 and E2F1. Furthermore, VCX treatment increased the formation of reactive oxygen species and the expression level of autophagy markers, Beclin 1 and LC3-II. Importantly, these cellular changes by VCX increased the chemo-sensitivity of MDA-MB-231 cells to doxorubicin. Discussion: The present study explores the molecular mechanisms of VCX-mediated inhibitory effects on the growth and proliferation of TNBC MDA-MB-231 cells through the induction of apoptosis, cell cycle arrest, and autophagy. The study also explores the role of BCL-2 as a novel targeted therapy for breast cancer.

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Background: Abnormal accumulation of senescent cells in the vessel wall leads to a compromised vascular function contributing to vascular aging. Soluble DPP4 (dipeptidyl peptidase 4; sDPP4) secretion from visceral adipose tissue is enhanced in obesity, now considered a progeric condition. sDPP4 triggers vascular deleterious effects, albeit its contribution to vascular aging is unknown. We aimed to explore sDPP4 involvement in vascular aging, unraveling the molecular pathway by which sDPP4 acts on the endothelium. Methods: Human endothelial cell senescence was assessed by senescence-associated β-galactosidase assay, visualization of DNA damage, and expression of prosenescent markers, whereas vascular function was evaluated by myography over human dissected microvessels. In visceral adipose tissue biopsies from a cohort of obese patients, we explored several age-related parameters in vitro and ex vivo. Results: By a common mechanism, sDPP4 triggers endothelial cell senescence and endothelial dysfunction in isolated human resistance arteries. sDPP4 activates the metabotropic receptor PAR2 (protease-activated receptor 2), COX-2 (cyclooxygenase 2) activity, and the production of TXA 2 (thromboxane A2) acting over TP (thromboxane receptor) receptors (PAR2–COX-2–TP axis), leading to NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3) inflammasome activation. Obese patients exhibited impaired microarterial functionality in comparison to control nonobese counterparts. Importantly, endothelial dysfunction in obese patients positively correlated with greater expression of DPP4, prosenescent, and proinflammatory markers in visceral adipose tissue nearby the resistance arteries. Moreover, when DPP4 activity or sDPP4-induced prosenescent mechanism was blocked, endothelial dysfunction was restored back to levels of healthy subjects. Conclusions: These results reveal sDPP4 as a relevant mediator in early vascular aging and highlight its capacity activating main proinflammatory mediators in the endothelium that might be pharmacologically tackled.

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Venetoclax (VCX), formally known as ABT‐199, is a selective and orally bioavailable small‐molecule B‐cell lymphoma 2 (BCL‐2) Homology 3 (BH3) mimetic, with potential pro‐apoptotic and antineoplastic activities. In April 2016, VCX was approved in the USA to treat patients with chronic lymphocytic leukemia (CLL) with chromosome 17p deletion who have received at least one prior treatment. VCX inhibits the activity of a novel target, Bcl‐2 protein, liberating the BH3‐only proteins and thus restoring apoptotic processes in tumor cells, via activation of BAK/BAX‐mediated apoptosis. The BCL‐2 protein is commonly expressed in several of human cancer including solid tumors, such as breast cancer, ovarian, lung, colorectal, stomach, and prostate cancers, as its expression has been shown to be associated with an aggressive disease course and poor survival. Although, BCL‐2 is expressed in several sub‐types of breast cancer, neither VCX nor any other BCL‐2 inhibitor agents alone or in combination with other chemotherapeutic agents have been tested against triple negative breast cancer (TNBC). Accordingly, we hypothesized that VCX is able to induce TNBC cell growth inhibition via induction of apoptosis, autophagy, and cell cycle arrest. To test this hypothesis, human TNBC MDA‐MB‐231 cells were treated for indicated intervals with increasing concentrations of VCX, thereafter, the effects of VCX on cell proliferation and cell cycle arrest were determined using flow cytometry. Furthermore, the apoptogenic effect of VCX was explored by measuring the percentage of apoptotic cells using flow cytometry and the expression of caspases, BAX, and BCl2 genes at the mRNA, protein, and activity levels. The results of the present study showed that VCX decreased the growth and proliferation of TNBC MDA‐MB‐231 cells in a concentration manner. This growth suppression was associated with a significant increase in the percentage of apoptotic MDA‐MB‐231 cells with a significant increase in the expression of several apoptotic genes, caspase3, caspase7, and BAX in concentration‐dependent manner. Importantly, the VCX‐induced‐apoptosis was accompanied with cell cycle arrest at G0/G1 phase with a concentration‐dependent inhibition of cell cycle proliferator genes, such as Cyclin D1 and E2F1. In addition, VCX treatment induced a significant increase in the level of autophagy marker LC3‐II mRNA and protein expression levels. In conclusion, the present study demonstrates the first evidence that VCX induces human TNBC MDA‐MB‐231 cell growth inhibition through the induction of apoptosis, cell cycle arrest, and autophagy mechanisms.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Introduction: Vascular aging is associated with endothelial cell senescence, favoring low-grade inflammation, endothelial dysfunction, and cardiovascular diseases. Cell senescence arises from a wide variety of endogenous and exogenous stressors including some anticancer agents such as doxorubicin. Recently, doxorubicin was linked to the innate immunity component NLRP3 inflammasome which is implicated in many vascular inflammatory disorders. There is a need for therapeutic tools to help cancer patients who have been exposed to cardiovascular toxic chemotherapy averting premature vascular complications. We investigated whether resolvin E1 (RvE1), an endogenous lipid mediator of the inflammation resolution phase, could prevent doxorubicin-induced senescence in cultured human umbilical veins endothelial cells (HUVEC) with focus on a potential involvement of the NLRP3 inflammasome. Materials and Methods: Cell senescence was quantified by senescence-associated-β-galactosidase (SA-β-gal) staining. The expression of senescence markers (γH2AX, p21, p53) and inflammatory markers (pP65, NLRP3) was determined via Western blot. NLRP3 inflammasome activation was determined by visualizing the formation of ASC specks by indirect immunofluorescence. Results: Doxorubicin (25 nmol/L) augmented the number of SA-β-gal positive HUVEC and the levels of γH2AX, p21 and p53 which were all reduced by RvE1 (10 nmol/L). In doxorubicin-treated cells, RvE1 further reduced the expression of pP65 and NLRP3 proteins and the formation of ASC specks as did the inflammasome assembly inhibitor MCC950 (1µmol/L). Additionally, both MCC950 and interleukin-1 receptor inhibitor anakinra diminished SA-β-gal positive staining induced by doxorubicin. Conclusion: RvE1 offers a novel therapeutic approach against doxorubicin-induced cardiovascular toxicity and subsequent age-related vascular disorders by counteracting endothelial senescence through the modulation of NLRP3- inflammasome activation.

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