in adenocarcinomic human alveol ar epithelial cells a549 ( a model for non - small cell lung cancer ), 50 μm and 150 μm ole - induced apoptosis after 24 h incubation was mediated by the decrement in bcl - 2 and bcl - xl anti - apoptotic proteins flanked by the increase in ( i ) mitochondrial - located pro - apoptotic protein bax, ( ii ) cytochrome c release from the mitochondria, ( iii ) activation of apoptosome component apoptotic protease activating factor - 1 ( apaf - 1 ), ( iv ) activation of caspase - 3, and ( v ) mitochondrial methylglyoxal detoxicating enzyme glo2 ( mglo2 ), which physically interacted with bax. consistent with these data, incubation of non - small cell lung cancer cell line h1299 with 50 – 200 μm ole for 24 h elicited a dose - dependent g2 / m phase cell cycle block and apoptosis, with effects on bcl - 2, bax, cytochrome c, and caspase - 3 that were similar to those documented for a549 cells. however, the underlying molecular mechanism ruling ole activity differed between the two cell lines. the effects elicited by 150 μm ole on mglo2 levels in a549 cells were strictly dependent on ole - induced increase in superoxide dismutase 2 ( sod2 ) detoxicating action against superoxide ( o2 ), and on the inhibition of the akt signalling pathway. this was not surprising, since in a549 cells, o2 supports akt activations, promoting cell survival. in h1299 cells, the observed apoptosis was instead determined by ole - induced phosphorylation of p38 mitogen - activated protein kinase ( mapk ), accompanied by an increased rate of phosphorylation of activating transcription factor - 2 ( atf - 2 ), involved in cell cycle regulation, as documented in both tumorigenesis and cell death, and the upregulation of genes ruling cell metabolism and apoptosis. in a549 cell line, ht showed an increase in effectiveness of its anti - proliferative activity with respect to time, with the ic50 values changing from 230. 60 μm to 149. 36 μm in 72 h. this piece of data was confirmed in another report. in fact, for a549 cells, mean ic50 ± s. e. m. = 147. 0 ± 16. 5 μm was reported after 48 h incubation. malignant pleural mesothelioma arises from mesothelial cells and is characterized by a pronounced aggressiveness and poor prognosis. on the basis of histological features, mesothelioma is classified as epithelioid, sarcomatoid, or biphasic, with epithelioid type offering the best median survival. pleural epithelioid mesothelioma ren cell line was utilized to demonstrate that ole exhibited a cytotoxic activity ( ic50 = 25 μg / ml, ≈46 μm ), and that both ole and ht ( 10 – 100 μm ) mobilized extracellular ca in a dose - dependent manner. breast cancer is the most frequent malignant tumor in women worldwide, with a constantly rising incidence. treatment of breast cancer is based on the molecular subtype, a classification that in the first instance takes into account the immunoh istochemically assayed expression of hormone receptors estrogen receptor ( er ) and progesterone receptor ( pr ), and gene amplification or overexpression of human epidermal growth factor receptor 2 ( her2 ). tripl e negative breast cancer identifies a category of tumors lacking the three mentioned receptors. in an in vivo model of tumor xenograft ( tripl e negative mda - mb - 231 cell line ) in balb / c olahsd - foxn1 mice, animals receiving 50 mg / kg ole for 4 weeks showed a decrease in tumor size, together with a reduction in actors involved in cell growth / proliferation: transcription factor nf - κb and cyclin d1. instead, levels of tumor suppressor p21 increased after ole injection. the effects elicited by ole were accompanied by the induction of apoptosis, as demonstrated by caspase - 3 activation, increase in bax levels, and reduction in bcl - 2 protein expression. in vitro, ole action appears to be independent of her2 gene amplification / overexpression and hormone receptor status. using mcf - 7 cell line, which is devoid of her - 2 overexpression, 200 μg / ml ( ≈370 μm ) ole for 48 h showed a specific cytotoxic effect on mcf - 7 cell line, leaving the non - cancerous cell line mcf - 10a unharmed. the observed effect on cell viability was accompanied by the upregulation of the expression of prdx1 - prdx6, encoding for antioxidant and chaperone proteins peredoxins. the induction of apoptosis triggered by the same concentration of ole in mcf - 7 cells was confirmed in another study after 12 h incubation. treatment of mcf - 7 cells with 200 μm and 400 μm ole for 24 h produced a notable decrease in cell viability. specifically, at a concentration of 400 μm, ole increased mcf - 7 cell death by apoptosis induction. ole effects might depend on the upregulation of p53 and bax, and downregulation of bcl - 2, as demonstrated by incubating mcf - 7 cells with 200 μm ole for 48 h. other studies using a very large concentration of ole ( 600 μg / ml, ≈1100 μm ) shed some light on a different mechanism ruling ole - dependent induction of apoptosis. in fact, treatment of mcf - 7 cells with the mentioned ole concentration for 48 and 72 h reduced histone deacetylase 2 ( hdac2 ) and hdac3 gene transcription in a time - dependent manner, and downregulated oncomirs mir - 21 and mir - 155. however, ole - mediated effects on er - positive cell viability may also be appreciated at lower doses. in fact, 30 μm and 50 μm ole were able to reduce mcf - 7 cell viability after 48 h, with no significant increase in the apoptotic rate. moreover, 150 μm ole reduced cell viability of er - positive mcf - 7 and t47d cells after 24 h. further experiments on mcf - 7 cell line demonstrated a dose - dependent ole - mediated antiproliferative effect on 17β - estradiol ( e2 ) - induced cell growth when ole concentration was used in the range of 10 – 75 μm. instead, concentrations ≥ 100 μm were found to be toxic. as regards a possible anti - estrogenic action, 10 μm ole had no effect on estrogen receptor α ( erα ) basal activation, and 10 – 75 μm ole had irrelevant activity on e2 - induced erα activation and e2 - modulated erα expression, but reduced e2 - induced erk1 / 2 phosphorylation. in er - positive cells, invasiveness may be suppressed by ole - mediated induction of autophagy. in fact, treatment of mcf - 7 and t47d cell lines with 100 μm ole reversed hepatocyte growth factor ( hgf ) - and 3 - methyladenine ( 3 - ma, an autophagy inhibitor ) - induced migration, upregulating lc3ii / lc3i and beclin1, while downregulating p62. ole was also able to reduce proliferation in tripl e negative breast cancer cell lines mda - mb - 231 and mda - mb - 468 after 48 h incubation ( ic50 = 500 μm ), with rna sequencing revealing alterations of the expression profile of genes involved in cell death, apoptosis, and response to stress. other reports documented that a dose as low as 50 μm was able to reduce mda - mb - 231 cell viability after 72 h incubation and ic50 = 36. 2 μm was estimated for 72 h treatment. a further attempt to deepen the mechanism of action in tripl e negative breast cancer showed that 12. 5 – 100 μm ole affected mda - mb - 231 cell viability in a dose - and time - dependent manner, reducing cellular migration and invasion capabilities, especially at doses ≥ 25 μm. ole was able to induce dose - dependent apoptosis after 72 h incubation. a deeper analysis, performed using a dose of 100 μm, revealed caspase - 3 / 7 activation after 48 and 72 h, and a reduction in nf - κb phospho - p65 nuclear localization after 12 h. in a breast cancer cell line identified by the authors only by the letters “ mda ”, 200 μg / ml ole was able to interfere with the metastatic process, causing a time - dependent increase in mrna levels of mmp inhibitors timp metallopeptidase inhibitor 1 ( timp1 ) and timp3, while timp4 showed no further increase after 48 h incubation. simultaneously, the same concentration of ole triggered a time - dependent decline in mmp2 and mmp9 mrna levels.