Gi Ming Lai

Basal levels and patterns of anticancer drug-induced activation of nuclear factor-κB (NF-κB), and its attenuation by tamoxifen, dexamethasone, and curcumin in carcinoma cells

Nuclear factor-kappaB (NF-kappaB) has been implicated in the development of drug resistance in cancer cells. We systematically examined the baseline levels of NF-kappaB activity of representative carcinoma cell lines, and the change of NF-kappaB activity in response to a challenge with four major anticancer drugs (doxorubicin, 5-fluorouracil, cisplatin, and paclitaxel). We found that the basal level of NF-kappaB activity was heterogeneous and roughly correlated with drug resistance. When challenged with various drugs, all the cell lines examined responded with a transient activation of NF-kappaB which then declined to basal level despite variation in the concentration of the agent and the timing of the treatment. In contrast to tumor necrosis factor-alpha (TNF-alpha), which activates NF-kappaB in minutes, NF-kappaB activation induced by anticancer drugs usually occurred more than 1hr after stimulation. A gradual increase of total NF-kappaB and its nuclear translocation, and cytoplasmic translocation of nuclear IkappaBalpha and its degradation were involved in this process. In particular, when cells were pretreated with common biologic modulators such as tamoxifen, dexamethasone, and curcumin, the doxorubicin-induced NF-kappaB activation was attenuated significantly. This inhibition may play a role in sensitizing cancer cells to chemotherapeutic drugs. This study has demonstrated that activation of NF-kappaB is a general cellular response to anticancer drugs, and the mechanism of activation appears to be distinct from that induced by TNF-alpha. These observations may have implications for improving the efficacy of systemic chemotherapy for cancer patients.

Receptor tyrosine kinase AXL is induced by chemotherapy drugs and overexpression of AXL confers drug resistance in acute myeloid leukemia

By using a novel profiling analysis of protein tyrosine kinases differentially expressed in the sensitive and refractory leukemia from the same patients we found that AXL was upregulated in drug-resistant leukemia. Furthermore, AXL could be induced by chemotherapy drugs in the acute myeloid leukemia U937 cells and this induction was dependent on the CCWGG methylation status of the AXL promoter. In U937 cells ectopically overexpressing AXL, addition of exogenous Gas6 induced AXL phosphorylation and activation of the Akt and ERK1/2 survival pathways. The Gas6-AXL activation pathway of drug resistance was associated with increased expression of Bcl-2 and Twist. These results show that upregulation of AXL by chemotherapy might induce drug resistance in acute myeloid leukemia in the presence of Gas6 stimulation.

Cytotoxic triterpenes from Antrodia camphorata and their mode of action in HT-29 human colon cancer cells.

Five lanostane (2, 3, 4, 6 and 8) and three ergostane-type (1, 5 and 7) triterpenes isolated from the fruiting bodies of Antrodia camphorata were evaluated for their in vitro cytotoxic data against various cancer cell types. The three zhankuic acids, 1, 5 and 7 displayed the most potent cytotoxic effect with an IC(50) value of 22.3-75.0microM. The compound 3 was selectively cytotoxic in three colon cancer cell lines (HT-29, HCT-116 and SW-480) and a breast cancer model (MDA-MB-231), whereas 8 only showed its cytotoxicity against MDA-MB-231. None of these isolates was toxic to mammary epithelial (MCF10A) and primary foreskin fibroblast (HS68) cells, two human normal cell lines. The compounds 1, 5 and 7 were also demonstrated to induce apoptosis in HT-29 and SW-480 cells, as confirmed by sub-G1 cell cycle arrest. In HT-29 cells, the expression of apoptosis-associated proteins poly-(ADP-ribose) polymerase cleavage, Bcl-2 and procaspase-3 were suppressed by compounds 1, 5 and 7. A mixture containing 4microM each of compounds 1, 5 and 7 also showed a synergistic cytotoxic effect in HT-29 cells.

Sulfasalazine suppresses drug resistance and invasiveness of lung adenocarcinoma cells expressing AXL.

Metastasis and drug resistance are the major causes of mortality in patients with non-small cell lung cancer (NSCLC). Several receptor tyrosine kinases (RTKs), including AXL, are involved in the progression of NSCLC. The AXL/MER/SKY subfamily is involved in cell adhesion, motility, angiogenesis, and signal transduction and may play a significant role in the invasiveness of cancer cells. Notably, no specific inhibitors of AXL have been described. A series of CL1 sublines with progressive invasiveness established from a patient with NSCLC has been identified that positively correlates with AXL expression and resistance to chemotherapeutic drugs. The ectopic overexpression of AXL results in elevated cell invasiveness and drug resistance. Nuclear factor-kappaB (NF-kappaB) signaling activity is associated with AXL expression and may play an important role in the enhancement of invasiveness and doxorubicin resistance, as shown by using the NF-kappaB inhibitor, sulfasalazine, and IkappaB dominant-negative transfectants. In the current study, sulfasalazine exerted a synergistic anticancer effect with doxorubicin and suppressed cancer cell invasiveness in parallel in CL1 sublines and various AXL-expressing cancer cell lines. Phosphorylation of AXL and other RTKs (ErbB2 and epidermal growth factor receptor) was abolished by sulfasalazine within 15 min, suggesting that the inhibition of NF-kappaB and the kinase activity of RTKs are involved in the pharmacologic effects of sulfasalazine. Our study suggests that AXL is involved in NSCLC metastasis and drug resistance and may therefore provide a molecular basis for RTK-targeted therapy using sulfasalazine to enhance the efficacy of chemotherapy in NSCLC.

Dramatic synergistic anticancer effect of clinically achievable doses of lovastatin and troglitazone

Lovastatin (an HMG‐CoA reductase inhibitor) and troglitazone (a PPAR‐γ agonist) have been intensively studied prospectively for their application in cancer treatment. However, clinical trials of lovastatin or troglitazone in cancer treatment resulted in only limited responses. To improve their efficacy, lovastatin and troglitazone have, respectively, been tried to combine with other anticancer agents with varied outcomes. In our study, we found a dramatic synergism between lovastatin and troglitazone in anticancer at clinically achievable concentrations. This synergism was found in far majority of cell lines tested including DBTRG 05 MG (glioblastoma) and CL1‐0 (lung). This amazing synergism was accompanied by synergistic modulation of E2F‐1 and p27Kip1, which were reported to mediate the anticancer activities of lovastatin and troglitazone, respectively, and other cell cycle regulating proteins such as CDK2, cyclin A and RB phosphorylation status. With this dramatic combination effect of lovastatin and troglitazone, a promising regimen of cancer therapy may be materialized in the future.

Honokiol-induced apoptosis and autophagy in glioblastoma multiforme cells

Honokiol, a hydroxylated biphenyl compound isolated from the Chinese herb Magnolia officinalis, has been reported to have anticancer activities in a variety of cancer cell lines. The present study aimed to evaluate the anticancer effect and possible molecular mechanisms of honokiol in a glioblastoma multiforme (GBM) cell line. The anticancer activities of honokiol were investigated in the DBTRG-05MG GBM cell line. The effect of honokiol on cell growth was determined using a sulforhodamine B assay. Flow cytometry and immunoblotting were used to measure honokiol-induced apoptosis (programmed cell death type I) and autophagy (programmed cell death type II). Honokiol was observed to reduce DBTRG-05MG cell viability in a dose-dependent manner. At a dose of 50 μM, honokiol markedly decreased the expression of Rb protein and led to the cleavage of poly(ADP-ribose) polymerase and Bcl-xL to promote apoptosis in the cancer cells. In addition, markers of autophagy, including Beclin-1 and LC3-II, were also significantly increased. In addition to apoptosis, honokiol was also able to induce autophagy in the DBTRG-05MG cells. The mechanisms that are responsible for the correlation between honokiol-induced apoptosis and autophagy require further investigation. Such efforts may provide a potential strategy for improving the clinical outcome of GBM treatment.

Honokiol Eliminates Human Oral Cancer Stem-Like Cells Accompanied with Suppression of Wnt/β-Catenin Signaling and Apoptosis Induction

Honokiol, an active compound of Magnolia officinalis, exerted many anticancer effects on various types of cancer cells. We explored its effects on the elimination of cancer stem-like side population (SP) cells in human oral squamous cell carcinoma SAS cells. The sorted SP cells possessed much higher expression of stemness genes, such as ABCG2, ABCC5, EpCAM, OCT-4, CD133, CD44, and β-catenin, and more clonogenicity as compared with the Non-SP cells. After 48 h of treatment, honokiol dose dependently reduced the proportion of SP from 2.53% to 0.09%. Apoptosis of honokiol-treated SP cells was evidenced by increased annexin V staining and cleaved caspase-3 as well as decreased Survivin and Bcl-2. Mechanistically, honokiol inhibited the CD44 and Wnt/β-catenin signaling of SP cells. The Wnt signaling transducers such as β-catenin and TCF-4 were decreased in honokiol-treated SP cells, while the β-catenin degradation promoting kinase GSK-3α/β was increased. Consistently, the protein levels of β-catenin downstream targets such as c-Myc and Cyclin D1 were also downregulated. Furthermore, the β-catenin-related EMT markers such as Slug and Snail were markedly suppressed by honokiol. Our findings indicate honokiol may be able to eliminate oral cancer stem cells through apoptosis induction, suppression of Wnt/β-catenin signaling, and inhibition of EMT.

The Synergistic Anticancer Effect of Troglitazone Combined With Aspirin Causes Cell Cycle Arrest and Apoptosis in Human Lung Cancer Cells

Troglitazone (TGZ) is a synthetic thiazolidinedione drug belonging to a group of potent peroxisome proliferator‐activated receptor γ (PPARγ) agonists known to inhibit proliferation, alter cell cycle regulation, and induce apoptosis in various cancer cell types. TGZ is an oral anti‐type II diabetes drug that can reverse insulin resistance. For more then 100 yr, aspirin, a nonselective cyclooxygenase (COX) inhibitor, has been successfully used as an anti‐inflammatory drug. Recently, Aspirin (ASA) and some other nonsteroidal anti‐inflammatory drugs (NSAIDs) have drawn much attention for their protective effects against colon cancer and cardiovascular disease; it has been observed that ASAs anti‐tumor effect can be attributed to inhibition of cell cycle progression, induction of apoptosis, and inhibition of angiogenesis. In this report we demonstrate for the first time that, when administered in combination, TGZ and ASA can produce a strong synergistic effect in growth inhibition and G1 arrest in lung cancer CL1‐0 and A549 cells. Examination by colony formation assay revealed an even more profound synergy. In Western blot, combined TGZ and ASA also could downregulate Cdk2, E2F‐1, cyclin B1, cyclin D3 protein, and the ratio of phospho‐Rb/Rb. Importantly, apoptosis was synergistically induced by the combination treatment, as evidenced by caspase‐3 activation and PARP cleavage. The involvement of PI3K/Akt inhibition and p27 upregulation, as well as hypophosphorylation of Rac1 at ser71, were demonstrated. Taken together, these results suggest that clinically achievable concentrations of TGZ and ASA used in combination may produce a strong anticancer synergy that warrants further investigation for its clinical applications.

Nonautophagic cytoplasmic vacuolation death induction in human PC-3M prostate cancer by curcumin through reactive oxygen species -mediated endoplasmic reticulum stress

The antiapoptotic and antiautophagic abilities of cancer cells constitute a major challenge for anticancer drug treatment. Strategies for triggering nonapoptotic or nonautophagic cell death may improve therapeutic efficacy against cancer. Curcumin has been reported to exhibit cancer chemopreventive properties. Herein, we report that curcumin induced apoptosis in LNCaP, DU145, and PC-3 cells but triggered extensive cytoplasmic vacuolation in PC-3M cells. Electron microscopic images showed that the vacuoles lacked intracellular organelles and were derived from the endoplasmic reticulum (ER). Moreover, curcumin-induced vacuolation was not reversed by an apoptosis- or autophagy-related inhibitor, suggesting that vacuolation-mediated cell death differs from classical apoptotic and autophagic cell death. Mechanistic investigations revealed that curcumin treatment upregulated the ER stress markers CHOP and Bip/GRP78 and the autophagic marker LC3-II. In addition, curcumin induced ER stress by triggering ROS generation, which was supported by the finding that treating cells with the antioxidant NAC alleviated curcumin-mediated ER stress and vacuolation-mediated death. An in vivo PC-3M orthotopic prostate cancer model revealed that curcumin reduced tumor growth by inducing ROS production followed by vacuolation-mediated cell death. Overall, our results indicated that curcumin acts as an inducer of ROS production, which leads to nonapoptotic and nonautophagic cell death via increased ER stress.

Oxidative stress enhances Axl-mediated cell migration through an Akt1/Rac1-dependent mechanism

Persistent oxidative stress is common in cancer cells because of abnormal generation of reactive oxygen species (ROS) and has been associated with malignant phenotypes, such as chemotherapy resistance and metastasis. Both overexpression of Axl and abnormal ROS elevation have been linked to cell transformation and increased cell migration. However, the relationship between Axl and ROS in malignant cell migration has not been previously evaluated. Using an in vitro human lung cancer model, we examined the redox state of lung adenocarcinoma cell lines of low metastatic (CL1-0) and high metastatic (CL1-5) potentials. Here we report that Axl activation elicits ROS accumulation through the oxidase-coupled small GTPase Rac1. We also observed that oxidative stress could activate Axl phosphorylation to synergistically enhance cell migration. Further, Axl signaling activated by H2O2 treatment results in enhancement of cell migration via a PI3K/Akt-dependent pathway. The kinase activity of Axl is required for the Axl-mediated cell migration and prolongs the half-life of phospho-Akt under oxidative stress. Finally, downregulation of Akt1, but not Akt2, by RNAi in Axl-overexpressing cells inhibits the amount of activated Rac1 and the ability to migrate induced by H2O2 treatment. Together, these results show that a novel Axl-signaling cascade induced by H2O2 treatment triggers cell migration through the PI3K/Akt1/Rac1 pathway. Elucidation of redox regulation in Axl-related malignant migration may provide new molecular insights into the mechanisms underlying tumor progression.