further evidence supports ht - dependent reduction in cell viability of and induction of apoptosis in ht - 29 cells also at higher concentrations ( 600 – 800 μm ), and one study documented ic50 = 750 μm for ht29 and ht29 – 19a cell lines. a more detailed analysis revealed that treatment of ht - 29 cells with 400 μm ht for 24 h produced s and g2 / m phase cell cycle arrest and induced apoptosis, flanked by apoptosis ruling events: loss of mitochondrial potential, decrease in anti - apoptotic bcl - 2 protein level, reduction in phosphorylation of bad, increase in pro - apoptotic bax and bak, cytochrome c release, and activation of caspase - 3. a similar s phase cell cycle arrest was also observed for 800 μm ht, and induction of apoptosis also took place after 24 h incubation of ht - 29 cells with 600 μm and 800 μm ht. a 16 h incubation with 400 μm ht triggered endoplasmic reticulum stress in ht - 29 cells, with activation of unfolded protein response, as indicated by the increase in the spliced form of x - box binding protein 1 ( xbp - 1 ) mrna, the upregulation of chaperone 78 kda glucose - regulated protein ( grp78 / bip ) at both mrna and protein levels, the transient increase ( after 2 h ) in phosphorylation of pkr - like er - associated kinase ( perk ) and translation initiation factor - 2 ( eif2a ) ( accounting for inhibition of protein synthesis ), and time - dependent increase in chop protein levels ( responsible for ros production and bcl - 2 downregulation ) and nadph oxidase 4 ( nox4 ). in the same cellular model, apoptosis seemed regulated by ht - mediated increase in the phosphorylated form of c - jun n - terminal kinase ( jnk ), which is a central player in establishing cell faith ( apoptosis vs. proliferation ). accordingly, the phosphorylated form of jnk target, c - jun, was also increased in ht - 29 cells as a response to treatment with ht. both jnk and c - jun phosphorylations seem to reach a peak after 30 min and 4 h of incubation. in addition, 400 μm ht induced activator protein 1 ( ap - 1 ) transcriptional activity ( responsible for the expression of cell cycle regulator genes ), promoted a dynamic phosphorylation of erk1 / 2 ( with two peaks of phosphorylation — after 30 min and 2 h of incubation — both followed by a reduction in phospho - erk1 / 2 levels ), and abolished akt phosphorylation. all ht - dependent effects producing apoptosis as a result seemed to be mediated by protein phosphatase 2a ( pp2a ). moreover, 400 μm ht reduced hif - 1α protein levels and augmented the protein levels of proliferation and inflammation modulator peroxisome proliferator - activated receptor γ ( pparγ ) after 48 h incubation; 400 μm ht also elicited an increase in p53 levels starting after 16 h and lasting for 48 h. similarly, 800 μm ht had analogous effects on hif - 1α and pparγ protein expression starting after 24 h of incubation, but did not change p53 levels. finally, treatment of ht - 29 cells with 200 μm ht caused mobilization of endoplasmic reticulum ca depots, but the contribution of this ionic species to ht - induced apoptosis / effects was not explained. in sw620 cells, ht reduced cell viability at all tested concentrations ( 10 – 100 μm ), with a mechanism related to a decrease in fasn transcription and activity after 72 h exposure. a detailed analysis of the cell cycle revealed that 10 – 50 μm ht produced s phase cell cycle arrest, with apoptosis induction when ht was used at a concentration of 100 μm. treatment of hct116 cell line with 100 – 300 μm ht and lovo cell line with 100 – 400 ht μm for 72 h led to a dose - dependent decrease in cell viability, with ic50 calculated as 92. 83 μm and 140. 8 μm for hct116 and lovo cells, respectively. cell cycle analysis revealed that 72 h incubation of hct116 and lovo cells with 0. 0154 mg / ml ( ≈100 μm ) ht and 0. 0231 mg / ml ( ≈150 μm ) ht, respectively, caused g2 / m cell cycle arrest and promoted apoptosis. as regards the role of ros in high-dose ht - mediated effects, a report documented a dose - dependent reduction in cell viability of sw480 ( mean ic50 ± s. d. = 82 ± 10 ) and hct116 ( mean ic50 ± s. d. = 55 ± 7 ) cells treated with 50 – 100 μm for 72 h, in conditions favoring the accumulation of extracellular h2o2. pancreatic cancer, or pancreatic ductal adenocarcinoma, arises from the malignant transformation of pancreatic ductal cells, and represents the third leading cause of cancer - related death worldwide, with extremely low 5 - year and overall survival rates. up to now, the study of ole and ht effects in pancreatic cancer in vivo has been limited. in an in vivo orthotopic model of pancreatic cancer, obtained by injecting mouse pancreatic cancer cells panc02 in c57bl / 6 mice, proved that 200 mg / kg ht for 10 days suppressed tumor growth and proliferation. these results seem to depend on the modulation of the tumor microenvironment, since ht was able to reduce the accumulation of myeloid - derived suppressor cells in lymphoid organs, bone marrow, and tumor tissues. results in vitro underline the diverse sensitivity that cell lines exhibit against ole - and ht - mediated effects on cell growth. neither ole nor ht exhibited a cytotoxic effect on pancreatic ductal adenocarcinoma cell lines bxpc - 3 and cfpac - 1 for doses up to 300 μm, but both were able to decrease cell viability of pancreatic ductal adenocarcinoma cell line mia paca - 2 ( ic50 = 150. 1 μm for ole and 75. 1 for ht ) after 72 h incubation. both 200 μm ole and 100 μm ht produced g2 phase cell cycle arrest after 24 h treatment. the 48 h incubation with the same concentrations of ole and ht led to caspase - 3 - mediated apoptosis. the observed effects may be dependent on ole - and ht - mediated upregulation of ap - 1 components c - jun and fos. similarly, treatment of pancreatic ductal adenocarcinoma cell line panc - 1 with 10 μm, 32 μm, 100 μm, and 320 μm ht for 24, 48, and 72 h reduced cell viability in a dose - and time - dependent fashion; doses as low as 19 μm were able to induce apoptosis ( as detected after 24 h ), with caspase - 9 and bax upregulation, and mmp - 2 and mmp - 9 downregulation at mrna levels after 72 h incubation. treatment of mouse pancreatic cancer cell line panc02 with 50 μm, 150 μm, and 200 μm ht for 48 h elicited a dose - dependent inhibition of cell proliferation and induction of apoptosis. further experimental evidence revealed that 100 μm ht inhibited the expression of phospho - stat3 and cyclin d1 after 24 h. cervical cancer is the fourth most common cancer among women worldwide, and the protagonist of an internationally shared effort towards prevention. cervical cancer cases may be classified into two major histological subtypes: squamous cell carcinoma and adenocarcinoma. in human cervical adenocarcinoma hela cells, 50 – 100 μm ole promoted the accumulation of s phase cells with only a modest increase in the percentage of sub - g1 cells, whereas 200 μm ole was able to induce g2 / m cell cycle arrest and apoptosis through the augmented expression of pro - apoptotic bax protein, the reduction in anti - apoptotic bcl - 2, the release of mitochondrial cytochrome c in the cytosol, and caspase - 9 mediated activation of caspase - 3. these effects were connected to ( i ) an increase in the phosphorylated form of apoptosis regulator jnk, which in turn ruled an increase in the phosphorylated forms of cell cycle controller activator protein 1 ( ap - 1 ) components c - jun and atf - 2, and ( ii ) an increase in the levels of tumor suppressors p53 and p21. another report documented the cytotoxic effect of 100 – 1200 μg / ml ( ≈185 – 2220 μm ) ole in hela cells after 24 and 48 h treatments in a dose - and time - dependent manner, with 24 h ic50 = 600 μg / ml and 48 h ic50 = 300 μg / ml. a 50 μg / ml, 100 μg / ml, 200 μg / ml, 400 μg / ml, and 600 μg / ml ole - dependent increase in the apoptotic cell rate was detected after 48 h incubation.