The roles of JNK and apoptotic signaling pathways in PEITC-mediated responses in human HT-29 colon adenocarcinoma cells.

Phenethyl isothiocyanate (PEITC) is a potential chemopre-ventive agent that is present naturally in widely consumedvegetables, especially in watercress. It has been extensivelyinvestigated for its anticancer activities against lung, fore-stomach and esophageal tumorigenesis. Here we investi-gated the pro-apoptotic effect of PEITC in HT-29 humancolorectal carcinoma cell line, and the mechanism of apop-tosis induced by PEITC. PEITC-induced apoptosis wasdetermined by DNA fragmentation assay and diamidino-2-phenylindole (DAPI) staining technique. To understand themechanisms of apoptosis induced by PEITC, we studied therole of caspases, mitochondria-cytochrome c release, andmitogen-activated protein kinase (MAPK) signaling path-ways involved in PEITC-induced apoptosis in HT-29 cells.Both the caspase-3 and -9 activities were stimulated byPEITC.Thereleaseofcytochromecfromthemitochondrialinter-space was time- and dose-dependent, with a maximalrelease at 50 mM after 10 h treatment. Three MAPKs [JNK(c-Jun N-terminal kinase), extracellular signal-regulatedprotein kinase (ERK) and p38 kinase] were activatedshortly after PEITC treatment in HT-29 cells. Importantly,the SP600125 compound, an anthrapyrazolone inhibitor ofJNK, but not the ERK and p38 inhibitor, suppressed apop-tosis induced by PEITC. Similarly, this JNK inhibitor at-tenuatedbothcytochromecreleaseandcaspase-3activationinduced by PEITC. In summary, this study shows thatPEITC can induce apoptosis in HT-29 cells in a time- anddose-dependent manner via the mitochondria caspasecascade,andtheactivationofJNKiscriticalfortheinitiationof the apoptotic processes. This mechanism of PEITC mayplay an important role in the killing of cancerous cells andoffer a potential mechanismfor itsanticanceraction invivo.IntroductionColon cancer is one of the leading causes of cancer death in theUS (1). Epidemiological studies have demonstrated an inverseassociation of colon cancer with intake of cruciferous vegeta-bles (2,3). Dietary isothiocyanates, such as PEITC, are presentin large quantities in cruciferous vegetables including water-cress, broccoli and cabbage (4). Many isothiocyanates areeffective chemopreventive agents against carcinogen-inducedcancers in experimental animals. Apart from the colorectum,isothiocyanates inhibit cancer formation in various tissues suchas rat lung, esophagus, mammary gland, liver, small intestineand bladder (5–9). Previous studies suggested that chemopre-ventive activity of isothiocyanates against chemically inducedcancers is due to their ability to induce Phase II drug meta-bolizing enzymes such as glutathione S-transferase and qui-none reductase, which detoxify the activated carcinogenicmetabolites (10,11). It has also been shown that certain iso-thiocyanates may inhibit enzymes such as cytochrome P-450involved in the bioactivation of carcinogens (12). Furthermore,previous studies suggested that isothiocyanates could induceapoptosis in certain types of cancer cells. For example, it hasbeen shown that PEITC and AITC (allyl isothiocyanate) caninduce apoptosis in human leukemia HL-60 cells (13,14).PEITC has also been shown to induce apoptosis in HeLacervical cancer cells and PC-3 prostate cancer cell line (15,16).The mitogen-activated protein (MAP) kinase family is acti-vated in response to a wide variety of extracelluar stimuli andmediates signal transduction cascades that play an importantregulatory role in cell growth, differentiation and apoptosis(17). In the mammalian system, the biochemical properties ofthree MAP kinases have been characterized in detail, theextracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK) also referred to as stress-activatedprotein kinase and the P38 MAP kinase (18). The ERK sub-group of MAP kinases is activated primarily by mitogenicstimuli such as growth factors (19). In contrast, the JNK andP38 pathways are activated primarily by a diverse array ofcellular stress including UV irradiation, hydrogen peroxide,DNA damage, heat and osmotic shock (18).It was reported that PEITC induced JNK activation in HeLaand HT-1080 cells (20). In Jurkat cells, PEITC was found toinduce a sustained activation of JNK1, and this was associatedwith the activation of MEKK1 (16). It was also reported that inp53-deficient PC-3 cells PEITC induced the activation of ERKand p38 but not JNK (21). In addition, Yang et al. (22) showedthat N-acetylcysteine conjugate of PEITC activated JNK, p38and ERK in benzopyrene-treated A/J mice.To better understand the anticancer mechanism of isothio-cyanates, we examined the effect of PEITC, a predominantisothiocyanate in cruciferous vegetables, on HT-29 humancolon carcinoma cells. Here, we report that PEITC is capableof inducing apoptosis by releasing cytochrome c from themitochondria and activation of caspase-9 and -3, and thismitochondrial caspase-mediated apoptosis is related to activa-tion of JNK.Materials and methods