miR‐181a induces sorafenib resistance of hepatocellular carcinoma cells through downregulation of RASSF1 expression

Sorafenib, a multi‐kinase inhibitor, is the only standard clinical drug for patients with advanced hepatocellular carcinoma (HCC); however, development of sorafenib resistance in HCC often prevents its long‐term efficacy. Therefore, novel targets and strategies are urgently needed to improve the antitumor effect of sorafenib. In the present study, we examined the novel mechanisms of sorafenib resistance of HCC cells by investigating the difference in sorafenib sensitivity between two HCC cell lines. Sorafenib induced more apoptosis of HepG2 cells compared to Hep3B cells. Sorafenib exposure to HepG2 cells but not Hep3B cells increased the expression of proapoptotic factor PUMA, and activated PARP and caspase‐3. Notably, microRNA‐181a (miR‐181a) expression levels were lower in HepG2 cells than in Hep3B cells. Exogenous miR‐181a expression in HepG2 cells reduced apoptosis, whereas inhibition of miR‐181a in Hpe3B cells increased apoptosis. In addition, we demonstrated that miR‐181a directly targets RASSF1, a MAPK signaling factor, and knockdown of RASSF1 increased sorafenib resistance. Taken together, these results suggest that miR‐181a provokes sorafenib resistance through suppression of RASSF1. Our data provide important insight into the novel therapeutic strategy against sorafenib resistance of HCC cells by targeting of miR‐181a pathway.     

Genistein synergizes with arsenic trioxide to suppress human hepatocellular carcinoma

Arsenic trioxide (ATO) is of limited therapeutic benefit for the treatment of solid tumors. Genistein exhibits anticancer and pro‐oxidant activities, making it a potential candidate to enhance the efficacy of ATO whose cytotoxicity is oxidation‐sensitive. This study sought to determine whether genistein synergizes with ATO to combat hepatocellular carcinoma (HCC). Three human HCC cell lines, namely HepG2, Hep3B, and SK‐Hep‐1, were incubated with ATO, genistein, or ATO + genistein. The cells were also pretreated with antioxidant agents N‐acetyl‐L‐cysteine (NAC) or butylated hydroxyanisole (BHA). Cell viability, apoptosis, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), expression of Bcl‐2, Bax, caspase‐9, and ‐3, and release of cytochrome c into the cytosol were examined. The synergistic effect of ATO and genistein was also assessed using HepG2 xenografts subcutaneously established in BALB/c nude mice. The results show that genistein synergized with ATO to reduce viability, induce apoptosis, and diminish the ΔΨm of cells. The combination therapy down‐regulated Bcl‐2 expression, up‐regulated Bax expression, enhanced the activation of caspase‐9 and ‐3, and increased the release of cytochrome c. The synergistic effect of ATO and genistein was diminished by pretreatment with NAC or BHA. Genistein increased the production of intracellular ROS, while ATO had little effect. Genistein synergized with a low dose of ATO (2.5 mg/kg) to significantly inhibit the growth of HepG2 tumors, and suppress cell proliferation and induce apoptosis in situ. There were no obvious side effects, as seen with a high dose of ATO (5 mg/kg). Combining genistein with ATO warrants investigation as a therapeutic strategy to combat HCC. (Cancer Sci 2010; 101: 975–983)     

Activation of AMP‐activated protein kinase by retinoic acid sensitizes hepatocellular carcinoma cells to apoptosis induced by sorafenib

To improve the outcome of cancer chemotherapy, strategies to enhance the efficacy of anticancer drugs are required. Sorafenib is the only drug to prolong overall survival of the patients with hepatocellular carcinoma (HCC), however, the outcome is still not satisfactory. Retinoids, vitamin A derivatives, have been known to exhibit inhibitory effects on various cancers including HCC. In this study, we investigated the effects of combined treatment using sorafenib and retinoids including all‐trans retinoic acid (ATRA), NIK‐333, and Am80 on HCC cells. Cell viability assays in six HCC cell lines, HepG2, PLC/PRF/5, HuH6, HLE, HLF, and Hep3B, revealed that 5 and 10 μM ATRA, concentrations that do not exert cytotoxic effects, enhanced the cytotoxicity of sorafenib, being much more effective than NIK‐333 and Am80. We found that ATRA induced AMP‐activated protein kinase activation, which was followed by reduced intracellular ATP level. Gene expression analysis revealed that ATRA decreased the expression of glycolytic genes such as GLUT‐1 and LDHA. In the combination treatment using ATRA and sorafenib, increased apoptosis, followed by the activation of p38 MAPK and JNK, the upregulation and translocation of Bax to mitochondria, and the activation of caspase‐3, was observed. Suppression of AMP‐activated protein kinase by siRNA restored the viability of the cells treated with ATRA and sorafenib. Our results thus indicate that ATRA is useful for enhancing the cytotoxicity of sorafenib against HCC cells by regulating the energy metabolism of HCC cells.     

Role of caveolin‐1 in hepatocellular carcinoma arising from non‐alcoholic fatty liver disease

The molecular features of hepatocellular carcinoma arising from non‐alcoholic fatty liver disease (NAFLD‐HCC) are not well known. In this study, we investigated the mechanism by which NAFLD‐HCC survives in a fat‐rich environment. We found that caveolin (CAV)‐1 was overexpressed in clinical specimens from NAFLD‐HCC patients. HepG2, HLE, and HuH‐7 HCC cell lines showed decreased proliferation in the presence of the saturated fatty acids palmitic acid and stearic acid, although only HLE cells expressed high levels of CAV‐1. HLE cells treated with oleic acid (OA) showed robust proliferation, whereas CAV‐null HepG2 cells showed reduced proliferation and increased apoptosis. CAV‐1 knockdown in HLE cells attenuated the OA‐induced increase in proliferation and enhanced apoptosis. Liquid chromatography–tandem mass spectrometry analysis revealed that the levels of OA‐containing ceramide, a pro‐apoptotic factor, were higher in HepG2 and CAV‐1‐deficient HLE cells than in HLE cells, suggesting that CAV‐1 inhibits apoptosis by decreasing the level of OA‐containing ceramide. These results indicate that CAV‐1 is important for NAFLD‐HCC survival in fatty acid‐rich environments and is a potential therapeutic target.     

Cardiac glycoside sensitized hepatocellular carcinoma cells to TRAIL via ROS generation,p38MAPK,mitochondrial transition,and autophagy mediation

A major concern in the clinical application of tumor necrosis factor related apoptosis‐inducing ligand (TRAIL) in tumors is the development of resistance. Therefore, agents that can potentially restore TRAIL sensitivity are important therapeutic targets for cancer treatment. Herein, we evaluated lanatoside c and digoxin, both of which are widely used cardiac glycosides (CGs), for their ability to sensitize human hepatocellular carcinoma cells (Huh‐7 and HepG2) through TRAIL‐induced apoptosis. CGs functionalize TRAIL as shown by its effect on intracellular reactive oxygen species (ROS) generation, which damages mitochondrial integrity and thereby confers intrinsic apoptotic caspase cascade during combined treatment. Caspase activation is dependent on ROS as shown by the ability of CGs to generate ROS and the ROS‐N‐acetylcysteine (NAC) relationship, which inhibits apoptosis during cotreatment by preventing the formation of caspase‐8 and ‐3. Furthermore, CGs triggered p38MAPK phosphorylation and NAC pre‐exposure blocked p38MAPK phosphorylation, which demonstrated that p38MAPK was dependent upon ROS generation. Additionally, CGs were found to be potent inducers of AMPK‐mediated protective autophagy as pharmacological and genetic autophagy inhibition reached the higher threshold of TRAIL‐mediated apoptosis. Finally, CGs downregulated the expression of the antiapoptotic protein Bcl‐2 and increased the translocation of proapoptotic protein cytochrome c, thereby inducing apoptosis. Collectively, these results indicate that CGs potentiate the enhanced cytotoxic capacity to TRAIL through ROS generation, p38MAPK phosphorylation, cell survival protein downregulation, and protective autophagy inhibition.     

Differential involvement of reactive oxygen species in apoptosis induced by two classes of selenium compounds in human prostate cancer cells

Selenium is a promising chemopreventive agent for prostate cancer, possibly via an induction of apoptosis. Earlier studies have shown that selenite induces DNA single strand breaks (SSBs), reactive oxygen species (ROS), p53 Ser‐15 phosphorylation and caspase‐dependent and ‐independent apoptosis, whereas a methylselenol precursor methylseleninic acid (MSeA) induces caspase‐mediated apoptosis regardless of p53 status. Here we address three main questions: What types of ROS are induced by selenite vs. MSeA in LNCaP (p53 wild type, androgen‐responsive) and DU145 (mutant p53, androgen‐independent) prostate cancer cells? Does ROS generation depend on androgen signaling? What are the relationships among ROS, DNA SSBs, p53 and caspases? We show that selenite (5 μM) induced superoxide and hydrogen peroxide in LNCaP cells much more than in DU145 cells and the ROS generation was not affected by physiological androgen stimulation. MSeA (10 μM) induced apoptosis without either type of ROS in both cell lines. In LNCaP cells, we established superoxide as a primary mediator for selenite‐induced DNA SSBs, p53 activation and caspase‐mediated apoptosis. Furthermore a p53‐dominant negative mutant attenuated selenite‐induced ROS, leading to a proportionate protection against apoptosis. The results support the p53‐mitochondria axis in a feedback loop for sustaining superoxide production to lead to efficient caspase‐mediated apoptosis by selenite. In contrast, caspase‐mediated apoptosis induced by MSeA does not involve ROS induction. Since p53 is frequently mutated or deleted in prostate cancer and many other cancers, our results suggest that genotoxic vs. nongenotoxic classes of selenium may exert differential apoptosis efficacy depending on the p53 status of the cancer cells. © 2007 Wiley‐Liss, Inc.     

Combination of proteasome and class I HDAC inhibitors induces apoptosis of NPC cells through an HDAC6‐independent ER stress‐induced mechanism

The current paradigm stipulates that inhibition of histone deacetylase (HDAC) 6 is essential for the combinatorial effect of proteasome and HDAC inhibitors for the treatment of cancers. Our study aims to investigate the effect of combining different class I HDAC inhibitors (without HDAC6 action) with a proteasome inhibitor on apoptosis of nasopharyngeal carcinoma (NPC). We found that combination of a proteasome inhibitor, bortezomib, and several class I HDAC inhibitors, including MS‐275, apicidin and romidepsin, potently induced killing of NPC cells both in vitro and in vivo. Among the drug pairs, combination of bortezomib and romidepsin (bort/romidepsin) was the most potent and could induce apoptosis at low nanomolar concentrations. The apoptosis of NPC cells was reactive oxygen species (ROS)‐ and caspase‐dependent but was independent of HDAC6 inhibition. Of note, bort/romidepsin might directly suppress the formation of aggresome through the downregulation of c‐myc. In addition, two markers of endoplasmic reticulum (ER) stress‐induced apoptosis, ATF‐4 and CHOP/GADD153, were upregulated, whereas a specific inhibitor of caspase‐4 (an initiator of ER stress‐induced apoptosis) could suppress the apoptosis. When ROS level in the NPC cells was reduced to the untreated level, ER stress‐induced caspase activation was abrogated. Collectively, our data demonstrate a model of synergism between proteasome and class I HDAC inhibitors in the induction of ROS‐dependent ER stress‐induced apoptosis of NPC cells, independent of HDAC6 inhibition, and provide the rationale to combine the more specific and potent class I HDAC inhibitors with proteasome inhibitors for the treatment of cancers.     

Apoptosis of liver cancer cells by vitamin K2 and enhancement by MEK inhibition

Vitamin K2 (VK2) is an anti-proliferative agent toward a variety of cancer including hepatocellular carcinoma (HCC). Because the growth inhibitory effect of VK2 to HCC has not been established yet, we investigated it in HCC cells in vitro. VK2 inhibited growth of Hep3B, but not of HepG2, HLF, and Huh6. VK2 induced the cell cycle arrest at the G1 phase and involvement of apoptosis was suggested because the sub-G1 fraction appeared in flow cytometric analysis and nuclear condensation and fragmentation appeared after VK2 treatment. VK2 activated extracellular signal-regulated kinase (ERK)1/2 in a mitogen-activated ERK-regulating kinase (MEK)-dependent manner in Hep3B and Huh6, but not in HepG2 and HLF. When ERK1/2 was inhibited by U0126, apoptosis by VK2 in Hep3B, but not in Huh6, was significantly enhanced. However, Western blot analysis revealed that neither apoptosis induction by VK2 nor enhancement of apoptosis by U0126 was mediated by caspase activation. These data demonstrated that VK2 induced apoptosis and activated the MEK/ERK1/2 signaling pathway in a cell-type specific manner, and a MEK inhibitor could augment the cell death in these cells.     

DNMT1 and DNMT3b silencing sensitizes human hepatoma cells to TRAIL‐mediated apoptosis via up‐regulation of TRAIL‐R2/DR5 and caspase‐8

DNA methylation plays a critical role in chromatin remodeling and gene expression. DNA methyltransferases (DNMTs) are hypothesized to mediate cellular DNA methylation status and gene expression during mammalian development and in malignant diseases. In this study, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b in cell proliferation and survival of hepatocellular carcinoma (HCC) cells. Gene silencing of both DNMT1 and DNMT3b by targeted siRNA knockdown reduces cell proliferation and sensitizes the cells to tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐mediated cell death. The proapoptotic protein caspase‐8 demonstrated promoter hypermethylation in HCC cells and was up‐regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. In addition, death receptor TRAIL‐R2/DR5 (TRAIL receptor 2/death receptor 5) did not exhibit promoter hypermethylation in HCC cells but was also up‐regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. Consistent with this observation, the combined transfection of DNMT1‐siRNA plus DNMT3b‐siRNA enhanced formation of the TRAIL‐death‐inducing signaling complex formation in HCC cells. In conclusion, our data suggest that DNA methylation of specific genomic regions maintained by DNMT1 and DNMT3b plays a critical role in survival of HCC cells, and a simultaneous knockdown of both DNMT1 and DNMT3b may be a novel anticancer strategy for the treatment of HCC. (Cancer Sci 2010)     

Apoptotic events induced by naturally occurring retinoids ATRA and 13-cis retinoic acid on human hepatoma cell lines Hep3B and HepG2

Two hepatoma cell lines were incubated for 72 h with ATRA and its analog 13cisRA and according to MTT assay, Hep3B cells were highly susceptible whereas HepG2 cells were more resistant to the treatment. At the high concentration of 166 microM, retinoids were able to induce apoptosis in both cell lines and the highest effect was observed in HepG2 cells treated with ATRA. TUNEL-based photometric ELISA showed that at the same retinoid concentration tested by flow cytometry, both cell lines showed apoptosis whereas plasma membranes were not significantly disrupted. Inhibitors of apoptosis Bcl-xL and survivin were downregulated in Hep3B cells by treatment with both retinoids. Bax, a pro-apoptotic protein, was not significantly upregulated in Hep3B cells, but was slightly increased in HepG2 cells treated with 13cisRA. Both procaspase-3 and procaspase-8 were cleaved in Hep3B cells, suggesting apoptosis could be triggered through the extrinsic pathway. In the case of HepG2 cells, lack of caspase activation suggests a mechanism dependent on other kind of proteases.     

Secretory clusterin contributes to oxaliplatin resistance by activating Akt pathway in hepatocellular carcinoma

Secretory clusterin (sCLU) is expressed in numerous cancers and is associated with the resistance to chemotherapy. However, the role of sCLU in the resistance of hepatocellular carcinoma (HCC) to oxaliplatin (OXA), a recently used third‐generation platinum agent, remains unclear. The stable transfectants that are depleted of or overexpress sCLU and OXA‐resistant cells were generated using human HCC cells. Overexpression of sCLU abrogated OXA‐induced inhibition of cell growth and cell apoptosis, but depletion of sCLU synergized with OXA to inhibit cell growth and enhance cell apoptosis, by regulating proteins involved in mitochondrial apoptosis pathways, such as Bcl‐2, Bax, Bcl‐xL and caspase‐9, and affecting phosphorylation of Akt and GSK‐3β. Overexpression of sCLU in either OXA‐resistant cells or stable transfectants that overexpress sCLU significantly increased phosphorylated Akt. However, specific inhibition of Akt enhanced sensitivity of sCLU‐overexpressing cells to OXA, but had no effect on sCLU expression, suggesting that the regulatory effects between sCLU and pAkt may be in a one‐way manner in HCC cells. The expression levels of sCLU affected the therapeutic efficacy of OXA to treat HCC tumors established in immunodeficiency mice. The results have demonstrated that sCLU contributes to OXA resistance by activating Akt pathway, indicating that sCLU may be a novel molecular target for overcoming OXA resistance in HCC.     

Flavonoids activated caspases for apoptosis in human glioblastoma T98G and U87MG cells but not in human normal astrocytes

BACKGROUND:

Human glioblastoma is a deadly brain cancer that continues to defy all current therapeutic strategies. The authors induced apoptosis in human glioblastoma T98G and U87MG cells after treatment with apigenin, (?)‐epigallocatechin, (?)‐epigallocatechin‐3‐gallate (EGCG), and genistein, which did not induce apoptosis in human normal astrocytes.

METHODS:

Induction of apoptosis was examined using Wright staining and ApopTag assay. Production of reactive oxygen species (ROS) and increase in intracellular free Ca²⁺ were measured by fluorescent probes. Analysis of mRNA and Western blotting indicated increases in expression and activities of the stress kinases and cysteine proteases for apoptosis. JC‐1 showed changes in mitochondrial membrane potential (ΔΨ_m), and use of specific inhibitors confirmed activation of kinases and proteases in apoptosis.

RESULTS:

Treatment of glioblastoma cells with apigenin, (?)‐epigallocatechin, EGCG, or genistein triggered ROS production that induced apoptosis with phosphorylation of p38 mitogen‐activated protein kinase (MAPK) and activation of the redox‐sensitive c‐Jun N‐terminal kinase 1 pathway. Pretreatment of cells with ascorbic acid attenuated ROS production and p38 MAPK phosphorylation. Increases in intracellular free Ca²⁺ and activation of caspase‐4 indicated involvement of endoplasmic reticulum stress in apoptosis. Other events in apoptosis included overexpression of Bax, loss of ΔΨ_m, mitochondrial release of cytochrome c and Smac into the cytosol, down‐regulation of baculoviral inhibitor‐of‐apoptosis repeat‐containing proteins, and activation of calpain, caspase‐9, and caspase‐3. (?)‐Epigallocatechin and EGCG also induced caspase‐8 activity. Apigenin, (?)‐epigallocatechin, EGCG, and genistein did not induce apoptosis in human normal astrocytes.

CONCLUSIONS:

Results strongly suggest that flavonoids are potential therapeutic agents for induction of apoptosis in human glioblastoma cells. Cancer 2010. © 2010 American Cancer Society.     

Ursolic acid induces PC‐3 cell apoptosis via activation of JNK and inhibition of Akt pathways in vitro

Ursolic acid (UA), a pentacyclic triterpenoid compound, has been demonstrated to have an antiproliferative effect in various tumors. We investigated the cell killing effects of UA in the human hormone refractory prostate cancer cell line, PC‐3 cells. Also, the molecular mechanisms underlying its antigrowth effect were explored. We found that UA treatment in vitro can effectively inhibit PC‐3 cell viability in a dose‐dependent manner by inducing apoptosis, demonstrated by annexin V‐FITC/propidium iodide staining. Both extrinsic and intrinsic apoptotic pathways appear to be triggered by UA treatment, because inhibiting activation of both caspase‐8 and ‐9 could prevent UA‐induced apoptosis in PC‐3 cells. The c‐Jun N‐terminal kinase (JNK) was found to be activated, followed by Bcl‐2 phosphorylation and activation of caspase‐9. On the other hand, UA inhibited the Akt pathway, subsequently upregulating the expression of Fas ligand (FasL), which initiates death receptor‐mediated apoptosis in PC‐3 cells. Importantly, experimentally lowering FasL expression by siRNA significantly inhibited UA‐induced caspase‐8 activation and at least partly attenuated the consequent apoptosis, suggesting an involvement of FasL and its regulating pathway in the cell killing effect of UA. UA also inhibited cell invasion by downregulating matrix metalloproteinase‐9 via inhibition of Akt in PC‐3 cells. Although further evaluation of the UA effects in vivo is needed, the present results suggest the potential utility of UA as a novel therapeutic agent in advanced prostate cancer. © 2010 Wiley‐Liss, Inc.     

Combined in vitro anti-tumoral action of tamoxifen and retinoic acid derivatives in hepatoma cells.

Chemotherapy does not have a prominent role in the treatment of hepatoma. However, an acyclic retinoid prevented tumor recurrence post-hepatectomy, and tamoxifen (TAM) induced apoptosis in tumor cells. Combination therapy of these agents on proliferation and apoptosis of hepatoma cells has not been explored. HepG2, Hep1B, Hepa1-6 and MH1C1 hepatoma cells were incubated with TAM, 9-cis- and all-trans retinoic acid (CRA, ATRA, respectively) alone or in combination. Proliferation rate was assessed and apoptosis was analyzed by flow cytometry, immunostaining, caspase activity assays and the expression of apoptosis- and/or cell cycle-related molecules. CRA and TAM, but not ATRA monotherapy were moderately effective. Apoptosis was accompanied by upregulation of caspase 3 and 8 activity, and increased p27, bax, caspase 3 expression, while the levels of p21cip/waf and bcl-2 were unchanged or decreased. Combination therapy enhanced apoptosis from a maximum of 60% after monotherapy to more than 90% after 96 h in all cell types. Pro-apoptotic effects were paralleled by inhibition of proliferation. Combination of TAM and CRA, but not ATRA, have an additive to synergistic anti-proliferative and pro-apoptotic effect on HCC cells. This justifies trials for HCC using combinations of these biological response modifiers.     

The dual PI3K/mTOR inhibitor NVP‐BEZ235 and chloroquine synergize to trigger apoptosis via mitochondrial‐lysosomal cross‐talk

On the basis of our previous identification of aberrant phosphatidylinositol‐3‐kinase (PI3K)/Akt signaling as a novel poor prognostic factor in neuroblastoma, we evaluated the dual PI3K/mTOR inhibitor BEZ235 in the present study. Here, BEZ235 acts in concert with the lysosomotropic agent chloroquine (CQ) to trigger apoptosis in neuroblastoma cells in a synergistic manner, as calculated by combination index (CI < 0.5). Surprisingly, inhibition of BEZ235‐induced autophagy is unlikely the primary mechanism of this synergism as reported in other cancers, since neither inhibition of autophagosome formation by knockdown of Atg7 or Atg5 nor disruption of the autophagic flux by Bafilomycin A1 (BafA1) enhance BEZ235‐induced apoptosis. BEZ235 stimulates enlargement of the lysosomal compartment and generation of reactive oxygen species (ROS), while CQ promotes lysosomal membrane permeabilization (LMP). In combination, BEZ235 and CQ cooperate to trigger LMP, Bax activation, loss of mitochondrial membrane potential (MMP) and caspase‐dependent apoptosis. Lysosome‐mediated apoptosis occurs in a ROS‐dependent manner, as ROS scavengers significantly reduce BEZ235/CQ‐induced loss of MMP, LMP and apoptosis. There is a mitochondrial‐lysosomal cross‐talk, since lysosomal enzyme inhibitors significantly decrease BEZ235‐ and CQ‐induced drop of MMP and apoptosis. In conclusion, BEZ235 and CQ act in concert to trigger LMP and lysosome‐mediated apoptosis via a mitochondrial‐lysosomal cross‐talk. These findings have important implications for the rational development of PI3K/mTOR inhibitor‐based combination therapies.     

Growth arrest induced by C75, A fatty acid synthase inhibitor, was partially modulated by p38 MAPK but not by p53 in human hepatocellular carcinoma

C75, a well-known fatty acid synthase (FAS) inhibitor, has been shown to possess potent anti-cancer activity in vitro and in vivo. In this study, we reveal that C75 is a cell cycle arrest inducer and explore the potential mechanisms for this effect in hepatocellular carcinoma (HCC) cell lines with abundant FAS expression: HepG2 and SMMC7721 cells with wt-p53, and Hep3B cells with null p53. The results showed FAS protein expression and basal activity levels were higher in HepG2 cells than in the other two HCC cell lines. Treatment with C75 inhibited FAS activity within 30 min of administration and induced G(2) phase arrest accompanied by p53 overexpression in HepG2 and SMMC7721 cells. By contrast, C75 triggered G(1) phase arrest in Hep3B cells, and RNA interference targeting p53 did not attenuate C75-induced G(2) arrest in HepG2 cells. Similarly, p53 overexpression via p53 plasmid transfection did not affect C75-induced G(1) phase arrest in Hep3B cells. However, we observed a clear correlation between p38 MAPK activation triggered by C75 and the induction of cell cycle arrest in all three HCC cells. Furthermore, treatment with the p38 MAPK inhibitor SB203580 reduced p38 MAPK activity and cell cycle arrest, and also partially restored cyclin A, cyclin B1, cyclin D1 and p21 protein levels. Collectively, it was p38 MAPK but not p53 involved in C75-mediated tumor cell growth arrest in HCC cells.     

α-TEA induces apoptosis of human breast cancer cells via activation of TRAIL/DR5 death receptor pathway

Vitamin E derivative RRR‐α‐tocopherol ether‐linked acetic acid analog (α‐TEA) induces apoptosis in MCF‐7 and HCC‐1954 human breast cancer cells in a dose‐ and time‐dependent manner. α‐TEA induces increased levels of tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and death receptor‐5 (DR5) and decreased levels of antiapoptotic factor, cellular FLICE‐like inhibitory protein (c‐FLIP L). DR5/TRAIL induced apoptosis involves downregulation of c‐FLIP (L), caspase‐8 activation, activated proapoptotic mediators tBid and Bax, mitochondrial permeability transition, and activation of caspase‐9. siRNA knockdown of either DR5 or TRAIL blocks the ability of α‐TEA to enhance DR5 protein levels, downregulate c‐FLIP(L) protein levels and induce apoptosis. Combination of α‐TEA + TRAIL acts cooperatively to induce apoptosis, and increase DR5 and decrease c‐FLIP (L) protein levels. siRNA knockdown of c‐FLIP produces a low level of spontaneous apoptosis and enhances α‐TEA‐ and TRAIL‐induced apoptosis. Taken together, these studies show that α‐TEA induces TRAIL/DR5 mitochondria‐dependent apoptosis in human breast cancer cells, and that TRAIL/DR5‐dependent increases in DR5 and decreases in c‐FLIP expression are triggered by TRAIL or α‐TEA treatments. © 2010 Wiley‐Liss, Inc.