after treating hepg2 cells with 50 μm ole for 24 h, the measured reduction in cell viability seemed related to ole - mediated accumulation of ros. however, one report documented the absence of effects on cell viability after treating hepg2 cells with 10 – 10 m ole for 24 h, and another study described no effect on hepg2 cell viability after 48 h incubation with 15 – 200 μm ole. the dose - dependent cytotoxic effect of ht on hcc was demonstrated in human hepatoma hepg2 and hep3b cell lines, with a time - dependent increase in effectiveness. in fact, 80 – 200 μm ht significantly affected cell metabolic activity after 48 h, whereas a concentration as low as 30 μm was sufficient to reduce the percentage of viable cells in both cell lines after 72 h of incubation. these effects might rely on inhibition of lipogenesis. in fact, in both cell lines ’ fatty acid synthase ( fasn ) activity was consistently inhibited by 200 μm ht ( with significant enzymatic inhibition starting with 30 μm and 80 μm ht for hepg2 and hep3b, respectively ), whereas farnesyl diphosph ate synthase ( fpps ) lipogenic enzyme activity was inhibited only in hepg2 cells. a further report confirmed the dose - and time - dependent inhibition of cell viability of human hcc cell lines hepg2, hep3b, and huh - 7, and ascitic fluid - derived cell line sk - hep - 1 for ht doses up to 400 μm, with g2 / m cell cycle arrest and inactivation of cell growth - promoting akt and nf - κb pathways. instead, the role of redox homeostasis in ht - dependent cytotoxicity is less clear in the context of hcc. cell antioxidant capacities were promoted by 48 h treatment with 80 – 200 μm ht in hepg2 cells; on the contrary, the same effect was elicited by only 48 h treatment with 30 μm ht in hep3b cells. however, ht triggers other molecular mechanisms that may influence hcc cell behavior besides affecting cell viability, at least in vitro. treatment of human hepatoma hepg2 cells with 50 – 200 μm ht determined an increase in intracellular ionized calcium levels ( [ ca ] i ) with the contribution of both ca influxes and mobilization of endoplasmic reticulum depots. anyway, ca dynamics seemed to be unrelated to ht - dependent reduction in cell viability, but they might be implicated in changes in the cell secretome, although definitive proofs are still missing. cholangio carcinoma is the term used to identify rare heterogeneous cancers arising in intrahepatic and extrahepatic bile ducts. instead, gallbladder cancer is the most common biliary tract cancer, characterized by a very unfavorable prognosis. treatment of cholangio carcinoma cell line kmbc and tfk - 1, and gallbladder carcinoma gbc - sd cells with 25 – 200 μm ht for 24, 48, and 72 h reduced cell proliferation in a time - and dose - dependent manner. a time - ( 24 – 72 h ) and dose ( 75 – 150 μm ) - dependent reduction in erk1 / 2 phosphorylation was detected in all the three mentioned cell lines. the 72 h incubation of kmbc, tfk - 1, and gbc - sd cells with 75 μm and 150 μm ht caused g2 / m phase cell cycle arrest, induced apoptosis, augmented the levels of cleaved parp, bax, cleaved caspase - 3, and cleaved caspase - 9, and reduced bcl - 2 levels. also, treatment of tfk - 1 cell xenograft tumor grown in nude balb / c mice with peritoneal injection of 500 mg / kg / day ht for 3 weeks reduced tumor growth in vivo. colorectal cancer, including tumors of the colon and / or rectum, is the third most common cancer and the second most common cancer - related cause of death worldwide. it is characterized by high rates of acquired multidrug resistance, leading to chemotherap y failure, relapse, and development of lethal disease. the classical dukes ’ classification disting uishes between “ type a ” and “ type b ” tumors, according to the absence or presence of tumor infiltration in extra - rectal tissues, respectively, and designates as “ type c ” those tumors with regional lymph node metastasis, and as “ type c1 ” and “ type c2 ” those malignancies exhibiting involvement of more distant lymph nodes. in an in vivo model of azoxymethane ( aom ) / dextran sulfate sodium ( dss ) - induced colorectal cancer in c57bl / 6 mice, 50 and 100 mg / kg ole reduced the incidence of colonic neoplasms, and levels of proliferation regulators nf - κb subunit p65, phosphorylated form of signal transducer and activator of transcription 3 ( stat3 ), and phospho - akt, while increasing bax protein expression. in addition, 100 mg / kg ole reduced cell proliferation ( as confirmed by reduced expression of proliferation marker ki67 ). colorectal carcinoma cell lines may exhibit different sensitivity towards ole - mediated effects on cell growth. in fact, treatment of colon carcinoma rko cell line with 20 – 80 μm ole diminished cell viability in a dose - dependent fashion. similarly, 10 – 100 μm ole reduced viability of dukes ’ type c colorectal adenocarcinoma sw620 cell line after 72 h incubation, becoming able to increase the rate of apoptosis at the highest concentration tested. on the contrary, 10 – 50 μm ole had no effect on the viability of colon adenocarcinoma ht29 cell line, but induced apoptosis only at a concentration of 100 μm. incubation with 400 μm and 800 μm ole reduced ht - 29 cell viability at all three tested time points ( 24, 48, and 72 h ), inducing g0 / g1 phase cell cycle arrest after 24 h treatment. however, only 800 μm ole was able to promote apoptosis in ht - 29 cells after 24 h incubation. treatment with 400 μm and 800 μm ole produced a reduction in protein expression of hif - 1α, which was detected after 2 h and persisted up to 48 h. instead, p53 protein expression was increased only by 48 h incubation with 800 μm ole. in vitro, the effects of low or nu trit ionally relevant ht doses are poorly explored, and ht is suspected to interfere with colorectal cancer insurgence acting as a methylation pattern modifying agent. in fact, incubation of human colorectal adenocarcinoma caco2 cell line with 10 μm ht increased dna methylation, leading to the repression of the crucial colorectal cancer promoter endothelin receptor type a ( ednra ). experimental evidence supports a pro - oxidant action of low doses of ht in colorectal cancer cells. treatment of human colorectal carcinoma hct116 cells and human dukes ’ type c colorectal adenocarcinoma sw620 cells with 5 – 20 μm ht for 24 h determined a raise in the apoptotic rates of both cell lines, ruled by a dose - dependent ros accumulation detected after 4 h of incubation, which in turn was caused by ht direct inhibition of thioredoxin ( trx ) reductase 1 ( trxr1 ), a key player in redox homeostasis. at higher concentrations, effects triggered by ht appear different and sometimes poorly reproducible. treatment of caco2 and ht - 29 cell lines with 100 μm ht for 8 h led to g1 phase cell cycle arrest, whereas extending the treatment for 48 h caused activation of caspase - 3. also, incubation of both cell lines with 150 μm for 8 h produced an increase in apoptosis rate, but ht contributed to the increase in the percentage of necrotic cells only in ht - 29 cells. after 24 and 48 h incubation, 6 ppm ( ≈39 μm ) ht was not sufficient to decrease ht - 29 cell viability, but after 72 h treatment, there was a significant cytotoxic effect, with ic50 = 12 ppm ( ≈78 μm ). at this concentration, ht elicited an increase in the transcription of tumor suppressor genes encoding for p21 and p27, while decreasing the expression of ccnd1 ( encoding for cyclin d1 ). after 24 h, 100 μm ht showed no effect on cell viability of ht - 29 cell line. however, 72 h incubation with 100 μm ht efficaciously reduced cell viability of and triggered apoptosis in ht - 29 cells. on the contrary, another report demonstrated a dose - dependent increase in the apoptotic rates of ht - 29, hct - 116, and lovo cells as well as dukes ’ type b colorectal carcinoma cell line sw480 for 24 and 48 h treatment with 100 – 400 μm ht.