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2′-Benzoyloxycinnamaldehyde (BCA), one of the derivatives of 2′-hydroxycinnamaldehyde (HCA) isolated from the bark of Cinnamomum cassia, induces apoptosis in human cancer cells. We found that BCA induces stronger antiproliferative effects in K-ras-transformed cells (RK3E-ras) than in isogenic non-transformed cells (RK3E). Treatment of RK3E-ras with BCA resulted in increased ROS generation and depletion of intracellular glutathione, whereas BCA-treated RK3E showed no significant increase in the ROS level with concurrent increase in intracellular glutathione (GSH). Thiol antioxidants recovered cell proliferation inhibition caused by BCA in both cell lines, while non-thiol antioxidants failed to recover cell death. BCA decreased metallothionein (MT) expression in RK3E-ras, while inducing remarkable MT expression in RK3E. The increase of intracellular GSH in RK3E is partially caused by differential induction of γ-glutamylcysteine synthetase (γ-GCS) due to BCA treatment. To evaluate the upstream pathway for differential expression of γ-GCS and MT, we analyzed early DJ-1 (PARK7) and NF-E2 p45-related factor 2 (Nrf2) changes after BCA treatment. In RK3E, DJ-1 expression considerably increased for 3 h with concurrent induction of Nrf2, whereas in RK3E-ras cells BCA decreased these protein levels. Based on these findings, it seems that the therapeutic selectivity of BCA in RK3E-ras results from decreased thiol antioxidants via decreased DJ-1 and Nrf2 expression. (Cancer Sci 2011; 102: 212–218)
2′-Benzoyloxycinnamaldehyde (BCA) (Fig. 1A) was origi-nally synthesized as a derivative of 2-hydroxycinnamaldehyde (HCA, Fig. 1B),(1) a natural compound isolated from the bark of Cinnamomum cassia Blume.(2) These compounds as antitumor agents induce apoptosis via reactive oxygen species (ROS) generation, caspase-3 activation(3) and the inhibition of proteasome activity.(4) BCA significantly blocked tumor growth in a nude mouse assay without bodyweight loss, making it a good drug candidate for cancer therapy.(3) It was also reported that the hepatic tumor volume and the total number of tumors decreased in BCA-treated mice compared with control H-ras12V transgenic mice.(5) In a rat oral tumor model induced by RK3E-ras-Fluc cells, direct injection of HCA into tumor significantly inhibited the growth of tumor mass.(6) Histological analysis also showed that HCA decreased tumor cell proliferation and induced apoptosis in a rat tumor.(6) When establishing a rat tumor model for the oral antitumorigenic test, we found that HCA and BCA had a greater antiproliferation effect in K-ras-transformed RK3E cells (RK3E-ras) than in isogenic normal K-ras cells (RK3E). We therefore tried to discern the underlying antitumor mechanism of BCA via the differential antiproliferative effect in these two cells.
K-ras is generally considered one of the most frequently mutated genes in a wide variety of human cancers. The highest incidences of K-ras mutation are found in: adenocarcinomas of the pancreas (90%), colon (50%) and lung (30%); in thyroid tumors (50%); and in myeloid leukemia (30%).(7) Therefore, using isogenic human cancer cells with or without the mutant K-ras gene could be a valuable strategy in drug screening for selective toxicity towards cancer cells.(8) A previous study showed that the intrinsic oxidative stress associated with K-ras-transformed oncogenic transformation may cause cancer cells that are highly dependent on their antioxidant systems to maintain redox balance.(9) In contrast, such stress is less likely in normal cells due to their low basal ROS level. This biochemical difference between normal and cancer cells may thus constitute a strategy for modulating cellular ROS to selectively kill cancer cells.(10) Hypothesizing that the ROS stress associated with oncogenic transformation would make cells highly dependent on their antioxidant systems to remove the damaging effect of ROS, we therefore compared intracellular ROS with major cellular antioxidant systems such as glutathione (GSH) and metallothionein (MT) levels, which are important in protecting against oxidative-stress-induced injury due to BCA treatment.
Nrf2 is generally known to regulate cytoprotective genes such as γ-glutamylcysteine synthetase (γ-GCS) and MT that contain an antioxidant response element (ARE) in their promoters. Another important mechanism for GSH regulation could be via the DJ-1 protein, which improves cell survival from oxidative damage by increasing cellular GSH levels.(11) Therefore, we evaluated whether BCA-induced differential antiproliferation in RK3E and RK3E-ras cells has been related with DJ-1 and Nrf2 pathways.
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Many anticancer agents can induce intracellular ROS to kill cancer cells. We have previously shown that BCA-induced apoptosis is completely blocked by pretreatment with GSH or NAC, concluding that intracellular ROS could be an important factor in apoptosis induction.(3) In the present study, we compared ROS production to BCA treatment to determine how BCA causes more effective cell death in K-ras-transformed cells than in normal K-ras cells. An increase in BCA-induced ROS in RK3E occurred after only 30 min and gradually decreased in a time-dependent manner, whereas BCA continuously increased the ROS generation in RK3-ras (Fig. 3A). We determined the effects of specific ETC. inhibitors on the ROS generation. Rotenone and/or KCN have been identified to inhibit the ROS generation caused by BCA in RK3E and RK3E-ras (Fig. 3C). Unexpectedly, rotenone and KCN failed to recover the antiproliferative effect of BCA, and only NAC restored cell proliferation completely in both cells (Fig. 4).
Many drug resistant cells are known to often show very low levels of ROS, usually due to high intracellular GSH levels and enhanced activities in antioxidant enzymes. A recent study showed that the electrophilic nature of cinnamaldehydes makes them potentially reactive with nucleophiles such as sulfhydryl groups in proteins and GSH.(13) Therefore, we measured the intracellular GSH levels upon BCA treatment. Interestingly, RK3E showed a continuous increase in intracellular GSH for 24 h, whereas the intracellular GSH level decreased early at 3 h in RK3E-ras, reaching a basal state at 16 h (Fig. 5A). Furthermore, pretreatment with BSO, an inhibitor of GSH synthesis, sensitized only RK3E to BCA (Fig. 6B). We tried to determine whether oxidative stress induced by hydrogen peroxide could also cause differential GSH depletion only in RK3E-ras. Hydrogen peroxide (1–100 mM) did cause oxidative stress, inducing concentration-dependent GSH depletion but in both cell lines (data not shown), suggesting that selective GSH depletion only in K-ras-transformed cells is specific to BCA.
A recent study showing BCA as an effective antitumor agent against hepatocellular carcinoma induced by the oncogenic H-ras12V gene,(14) confirmed that the MT gene was more than halved by BCA treatment. Metallothionein, which contains the highest amount of thiol groups within the cytoplasm, was initially identified as an antioxidant. Mechanisms underlying its function may include the maintenance of GSH levels.(15) Furthermore, MT thiol groups are able to bind several cytotoxic agents.(16) Comparing MT expression in both cells upon BCA treatment, we found the basal level of MT expression in RK3E to be lower than in RK3E-ras, while MT expression of RK3E exceeded that of RK3E-ras upon BCA treatment for 24 h (Fig. 5B).
These results led us to speculate that GSH and MT decreases in RK3E-ras can increase intracellular ROS, but the increased ROS is not critical for BCA-induced antiproliferation in RK3E-ras. To further verify the contribution of the intracellular ROS generation and GSH depletion to BCA-induced apoptosis, various thiol- and non-thiol antioxidants were pretreated with BCA. Thiol-antioxidants completely blocked apoptosis in both cells (Fig. 7). However, pretreatment with other non-thiol antioxidants were ineffective in preventing cell death. In addition to BCA’s differential antiproliferation in K-ras-transformed cells through intracellular GSH depletion, these data support the theory that intracellular thiol status and redox balance could be crucial in BCA-induced cell death. Many cancer therapeutic drugs function by depleting the intracellular thiol buffer system and thus disrupting the redox balance.(17,18) What is exciting about these studies is that GSH depletion occurs only in tumor cells enhancing antitumor cytotoxicity without increasing toxicity to normal tissue.(19) It is well known that intracellular redox status plays an important role in apoptosis, and as the most abundant non-protein antioxidant in the cell, intracellular GSH depletion often occurs at the onset of apoptosis.(20)
From the results of the present study, it can be suggested that the intracellular status of GSH affects toxicity of BCA in RK3E-ras. That is, increased levels of thiol antioxidants such as GSH and MT in RK3E are thus one mechanism of its resistance to BCA, as cytoplasmic binding of these antioxidants with BCA prevents the active molecules from reaching their target. However, as shown in the chromatogram of BCA mixed with GSH in vitro,(13) not all BCA compounds have been changed to BCA-GSH adduct even though there are enough GSH molecules for a reaction. These data partially resolve the inconsistency between intracellular GSH and the cell viability of RK3E after BCA treatment (Fig. 6). Furthermore, more specific mechanisms for therapeutic selectivity could be deduced from the activation process of ras by farnesylation of their cys residues. It is known that ras protein is farnesylated on the carboxy-terminal cysteine for activation, and that HCA inhibits farnesyl transferase in an enzymatic assay.(2) Therefore, further studies are needed to uncover whether BCA can inhibit ras activation via its reactivity with the sulfhydryl groups of the cys residues.
Endogenous GSH is synthesized from its constituent amino acids and γ-GCS is the rate-limiting step in de novo GSH synthesis. While anticancer drugs such as cisplatin and doxorubicin have been widely used to treat various tumors, Yao et al.(21) found γ-GCS expression to be elevated in cisplatin-resistant cancer cells. In the present study, BCA increased the γ-GCS protein level only in RK3E, with a remarkable decrease in RK3E-ras (Fig. 8). Therefore, the differential γ-GCS expression level between these two cells might be a key reason for why BCA causes significantly less cell death in RK3E.
Intracellular GSH upregulation could be via the DJ-1 protein, which improves cell survival from oxidative damage.(11) Interestingly, DJ-1 showed a cooperative transforming activity with H-ras in NIH3T3 cells.(22) Although the exact physiological role of DJ-1 is presently unclear, DJ-1 may mediate its antioxidant effects by Nrf2 pathway activation, which induces its target genes such as γ-GCS and MT.(23,24) In the present study, BCA increased DJ-1 and Nrf2 expression after only 30 min in RK3E (Fig. 9), possibly rendering upregulation of intracellular GSH via increased γ-GCS and MT expression. On the contrary, BCA decreased the expression of these proteins in RK3E-ras.
One of our further studies would be to confirm the differential effect of BCA in other normal/K-ras-mutated cells. We analyzed this effect in MCF-7 (normal K-ras) and MDA-MB-231 (K-ras-mutant) breast cancer cells. As expected and confirming our current results, BCA treatment induced stronger antiproliferation in MDA-MB-231 (IC50 28 μM) than in MCF-7 cells (IC50 > 60 μM) (data not shown).
Using isogenic cell lines, we showed that K-ras-transformed cells are more sensitive to BCA, which selectively decreases GSH and MT expression via downregulation of DJ-1. Increased levels of thiol antioxidants in RK3E are thus one mechanism of resistance to BCA (Fig. 10). More studies are necessary to directly evaluate in vivo BCA-GSH adduct formation in both cells. Furthermore, the differential regulation of DJ-1 expression and the role of DJ-1 in inactivating K-ras-mediated signals upon BCA treatment need to be elucidated.
Figure 10. Schematic representation showing the signaling pathway of the differential action of 2′-benzoyloxycinnamaldehyde (BCA) on normal and K-ras-transformed cells. γ-GCS, γ-glutamylcysteine syn-thetase; Nrf2, NF-E2 p45-related factor 2.
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