as regards ht activity in vivo, in a model of dimethylbenz [ α ] anthracene - induced mammary tumors in sprague – dawley rats treated with ht ( 0. 5 mg / kg, 5 days / week for 6 weeks ) reduced tumor growth, modulating the expression of genes involved in apoptosis and cell proliferation / survival. the relation between ht cytotoxic action in vitro and her2 overexpression / hormone receptor status is less clear. incubation of mcf - 7 cell line with 50 μg / ml ht ( ≈324 μm ) for 12 h was sufficient to trigger apoptosis, despite the fact that this result was not uniformly reproduced. in other experimental settings involving mcf - 7 cells, the anti - proliferative activity of ht became evident at higher as well as even lower concentrations. ht used in the range of 5 – 400 μm for 16 h had no effect on mcf - 7 cell proliferation; it became inhibited only at 600 μm. another report documented a dose - and time - dependent effect of ht on mcf - 7 cell viability, with 250 μm decreasing the percentage of viable cells after 72 h treatment, and 400 μm ht reducing cell viability as a consequence of 48 and 72 h incubation. however, at sub - lethal concentration ( 200 μm ), ht had an impact on both ( i ) oxidative stress response, augmenting mrna and protein levels of transcription factor nuclear respiratory factor 2 ( nrf2 ) and upregulating the transcription of its targets glutathione s - transferase alpha 2 ( gsta2 ) and heme oxigenase - 1 ( ho - 1 ), and ( ii ) energy homeostasis, reducing mrna level of mitochondria biogenesis regulator pparγ coactivator - 1α ( pgc - 1α ), while increasing its protein level and downregulating expression ( in terms of mrna ) of mitochondrial function regulators estrogen - related receptor α ( errα ) and deacetylase sirtuin 3 ( sirt3 ). other research records showed that a reduction in er - positive cell viability may be obtained even at lower ht doses, as demonstrated by 72 h treatment with 50 μm and 100 μm ht, which significantly diminished the percentage of viable mcf - 7 cells, and by 100 μm and 150 μm ht - induced reduction in mcf - 7 and t47d cell viability after 24 h. incubating mcf - 7 cells with 10 – 75 μm ht showed an inhibitory effect on e2 - induced cell growth, with ht becoming toxic at concentrations ≥ 100 μm. erα basal activation was induced by 10 μm ht, but ( similarly to ole ) when ht was used at concentrations of 10 – 75 μm, it exhibited no effect on e2 - induced erα activation and e2 - modulated erα expression, while reducing levels of phospho - erk1 / 2 ( perk1 / 2 ). as regards invasiveness of er - positive cells, experimental evidence obtained in mcf - 7 and t47d cells confirmed that 50 μm ht elicited effects similar to those recorded for ole on hgf - and 3 - ma - induced cell migration. in human tripl e negative breast cancer cells mda - mb - 231, ht seemed to lose effectiveness with respect to time, with ic50 values changing from 107. 17 μm to 183. 65 μm in 72 h. for the same cell line, higher ic50 values ( 230 μm ) were reported for 72 h treatment. on the contrary, another study demonstrated that 100 μm ht promoted mda - mb - 231 cell viability during the first 24 h, becoming ineffective after 48 and 72 h, whereas 250 μm ht became able to reduce cell viability after 72 h treatment, and 400 μm ht significantly diminished cell viability at all the three assayed time points ( 24, 48, and 72 h ). this piece of data is openly conflicting with reports documenting a loss of mda - mb - 231 viability after 72 h incubation at all tested ht doses ( 10 – 100 μm ). the reduction in tripl e negative breast cell viability may depend on ht acting as a copper chelator, thus perturbating copper homeostasis, as demonstrated in mda - mb - 231 cells by 100 μm ht - mediated increase in the copper chaperone for superoxide dismutase ( ccs ) after 48 h, and reduction in the subunit ii of the complex iv of the mitochondrial respiratory chain cytochrome c oxidase ( cco ) after 72 h. also, ht altered epithelial and mesenchymal markers, and reduced mda - mb - 231 aggressiveness and migration by diminishing copper - dependent akt phosphorylation. similar conclusions were drawn for mda - mb - 468 cells. other tripl e negative breast cancer cell lines were identified as particularly resistant towards ht - mediated cytotoxicity, e. g., sum159 ( ic50 = 300 μm ). in naturally her2 - overexpressing human breast cancer cells skbr3, ht concentrations up to 100 μm failed to elicit a cytotoxic response after 5 days, and only weakly interfered with cell proliferation, but reduced her2 protein expression after 48 h. however, in engineered her2 - overexpressing mcf - 7 cells, 100 μm ht efficaciously triggered apoptosis, diminished cell proliferation, and downregulated her2. other authors suggest that in vitro cytotoxic action of ole and ht may depend on cell density in culture, hypoxia, and ros homeostasis. han et al. observed that incubation with 200 μg / ml ole and 50 μg / ml ht for 12 h exhibited the most efficient inhibition of cell growth when mcf - 7 seeded cell number did not exceed 2 × 10 cell / well in a 96 - well plate. hypoxia increased ht cytotoxicity in mcf - 7 cell line, an effect that became evident at 400 μm ( vs. 600 μm in normoxic conditions ). in hypoxic conditions, 200 μm ht was also able to exert the same actions on oxidative stress response and energy homeostasis as those reported above for normoxic mcf - 7 cells. moreover, in hypoxic mcf - 7 cells, 75 – 200 μm ht reduced the amount of parp - 1, a dna - binding protein involved in oxidative stress response, but inhibition of parp - 1 activity was achieved only with 200 μm ht. the same concentration of ht accounted for the reduction in the phosphorylated form of kinase mammalian target of rapamycin ( mtor ), which in turn produced a reduction in hypoxia inducible factor - 1α ( hif - 1α ), one of the two subunits of the heterodimeric transcription factor hif - 1, ruling the cellular response to hypoxia. more importantly, 200 μm ht upregulated the transcription of angiogenic factors adrenomedullin ( am ) and vegf with an hif - 1α - independent mechanism. treatment of mda - mb - 231 cells with 200 μg / ml olive leaf extract containing 87 % ole for 24 h induced s phase cell cycle arrest and apoptosis by a mechanism that was strongly dependent on ole - mediated ros accumulation and downregulation of protein expression of catalase ( cat ) and sod2. treatment of mcf - 7 cells and another breast cancer cell line generically indicated by the authors simply as “ mda ” with 25 – 100 μm ht for 72 h reduced cell viability in a dose - dependent fashion ( mean ic50 ± s. d. = 52 ± 4 μm for of mda and 58 ± 8 μm for mcf - 7 ), mainly in culture conditions favoring h2o2 accumulation. ht may also have a role in changing the tumor microenvironment. aged quiescent normal human fibroblasts were able to stimulate mda - mb - 231 proliferation by synthetizing and secreting large amounts of chemok ine c - c motif ligand 5 ( ccl5 ), which in turn activated pro - proliferative erk1 / 2 and cyclin d1 signalling pattern. treatment of 100 μm and 200 μm ht prevented ccl5 accumulation in aged quiescent normal human fibroblasts, limiting the proliferation of mda - mb - 231 cells. a similar growth inhibition was also obtained for mcf - 7 cells. hepatocellular carcinoma ( hcc ) or hepatoma is the most frequent primary liver tumor and the sixth most common cancer worldwide, with a very modest survival rate and a complex management. results on ole - mediated effects on hepatoma cells are conflicting. in vitro, 20 – 80 μm ole reduced hepatoma huh7 and hepg2 cell line viability in a dose - dependent manner in 24 h. as determined in hepg2 cells, ole effects on cell viability are linked to apoptosis induction, mediated by an increase in bax and cleaved ( i. e., activated ) caspase - 9, caspase - 8, caspase - 3, and parp - 1 levels, and a decrease in phospho - akt and bcl - 2 protein levels.