And are expressed as of control. **, p,0.01 vs. control. doi:10.1371/journal.pone.0060065.gsenescence can be achieved at much lower doses of 
chemotherapy than those required to MK8931 web induce apoptosis, indicating that high doses of anticancer agent 22948146 may cause apoptosis whereas low level treatments primarily induce senescence in cancer cells [12]. Compared to the traditional 
apoptosis inducing strategies, this low dose approach can significantly reduce the side effects of anticancer therapy and thus improve the quality of life for cancer patients. Therefore, it is important to understand whether low dose RV can suppress lung cancer cell growth via the induction of premature senescence. RV is a nontoxic natural polyphenolic compound found in abundance in grapes, red wines, mulberries and other edible plants. Recent clinical trials have indicated that RV is well tolerated and relatively safe for use in humans [5?]. RV has been shown to inhibit the proliferation of a variety of cancer cells via buy 76932-56-4 inactivation of various cell survival pathways including the PI3kinase/AKT pathway [24]. RV also has been shown to exhibit potential chemopreventive activity in several carcinogen-induced tumor models including breast, intestine, liver, esophagus and colon [3,25,26]. Moreover, it has been reported that RV treatment inhibits pancreatic cancer growth and enhances the anticancer effects of gemcitabine possibly via suppression of NFB activation and down-regulating the expression of cyclin D1, COX-2, ICAM-1, MMP-9 and survivin in tumor tissues [27]. Although previous studies have indicated that RV, at high doses, can inhibit the proliferation of cancer cells by inducing apoptosis [28?1], a major challenge for the use of this CAM is that the concentration of RV required to induce apoptosis in tumor cells in vitro is too great to achieve in vivo in a clinical setting [5?,32]. Given that induction of senescence requires a much lower concentration of anticancer agents and thus produces fewer unwanted side effects [12], we sought to investigate if low dose RV treatment could inhibit the growth of cancer cells through the induction of premature senescence. In the present study, we show that low dose RV treatment leads to a significant increase in senescence-associated b alactosidase (SA-b-gal) staining and elevated p53 and p21 expression in NSCLC cells, suggesting that the anticancer effect of RV is largely attributable to the induction of senescence in lung cancer cells. Mechanistic studies reveal that RV-induced senescence is associated with increased DNA DSBs and ROS production in lung cancer cells. Moreover, our data also show that inhibition of ROS production by NAC attenuates RVinduced DNA DSBs and premature senescence. Altogether, these findings demonstrate that low dose RV treatment causesResveratrol-Induced Senescence in Cancer Cellspremature senescence in lung cancer cells via ROS-mediated DNA damage, which highlight a significant contribution of senescence induction to RV’s anticancer effects.Results RV inhibits the growth of lung cancer cells in a dosedependent mannerPrevious studies have indicated that higher doses of RV treatment may inhibit the proliferation of tumor cells by inducing apoptosis [28?1], but a major challenge for this apoptosis-causing strategy is that the concentration required to induce apoptosis in tumor cells in vitro is not reachable in vivo [5?,32]. Therefore, it is important to determine if low dose RV treatment affects the growth.And are expressed as of control. **, p,0.01 vs. control. doi:10.1371/journal.pone.0060065.gsenescence can be achieved at much lower doses of chemotherapy than those required to induce apoptosis, indicating that high doses of anticancer agent 22948146 may cause apoptosis whereas low level treatments primarily induce senescence in cancer cells [12]. Compared to the traditional apoptosis inducing strategies, this low dose approach can significantly reduce the side effects of anticancer therapy and thus improve the quality of life for cancer patients. Therefore, it is important to understand whether low dose RV can suppress lung cancer cell growth via the induction of premature senescence. RV is a nontoxic natural polyphenolic compound found in abundance in grapes, red wines, mulberries and other edible plants. Recent clinical trials have indicated that RV is well tolerated and relatively safe for use in humans [5?]. RV has been shown to inhibit the proliferation of a variety of cancer cells via inactivation of various cell survival pathways including the PI3kinase/AKT pathway [24]. RV also has been shown to exhibit potential chemopreventive activity in several carcinogen-induced tumor models including breast, intestine, liver, esophagus and colon [3,25,26]. Moreover, it has been reported that RV treatment inhibits pancreatic cancer growth and enhances the anticancer effects of gemcitabine possibly via suppression of NFB activation and down-regulating the expression of cyclin D1, COX-2, ICAM-1, MMP-9 and survivin in tumor tissues [27]. Although previous studies have indicated that RV, at high doses, can inhibit the proliferation of cancer cells by inducing apoptosis [28?1], a major challenge for the use of this CAM is that the concentration of RV required to induce apoptosis in tumor cells in vitro is too great to achieve in vivo in a clinical setting [5?,32]. Given that induction of senescence requires a much lower concentration of anticancer agents and thus produces fewer unwanted side effects [12], we sought to investigate if low dose RV treatment could inhibit the growth of cancer cells through the induction of premature senescence. In the present study, we show that low dose RV treatment leads to a significant increase in senescence-associated b alactosidase (SA-b-gal) staining and elevated p53 and p21 expression in NSCLC cells, suggesting that the anticancer effect of RV is largely attributable to the induction of senescence in lung cancer cells. Mechanistic studies reveal that RV-induced senescence is associated with increased DNA DSBs and ROS production in lung cancer cells. Moreover, our data also show that inhibition of ROS production by NAC attenuates RVinduced DNA DSBs and premature senescence. Altogether, these findings demonstrate that low dose RV treatment causesResveratrol-Induced Senescence in Cancer Cellspremature senescence in lung cancer cells via ROS-mediated DNA damage, which highlight a significant contribution of senescence induction to RV’s anticancer effects.Results RV inhibits the growth of lung cancer cells in a dosedependent mannerPrevious studies have indicated that higher doses of RV treatment may inhibit the proliferation of tumor cells by inducing apoptosis [28?1], but a major challenge for this apoptosis-causing strategy is that the concentration required to induce apoptosis in tumor cells in vitro is not reachable in vivo [5?,32]. Therefore, it is important to determine if low dose RV treatment affects the growth.
