Sensitization of cancer cells to paclitaxel-induced apoptosis by canagliflozin.

Cancer cells consume more glucose and usually overexpress glucose transporters which have become potential targets for the development of anticancer drugs. It has been demonstrated that selective SGLT2 inhibitors, such as canagliflozin and dapagliflozin, display anticancer activity. Here we demonstrated that canagliflozin and dapagliflozin synergistically enhanced the growth inhibitory effect of paclitaxel in cancer cells including ovarian cancer and oral squamous cell carcinoma cells. Canagliflozin also inhibited glucose uptake via GLUTs. The combination of paclitaxel and WZB117, a GLUT inhibitor, exhibited a strong synergy, supporting the notion that inhibition of GLUTs by canagliflozin may also account for the synergy between canagliflozin and paclitaxel. Mechanistic studies in ES-2 ovarian cancer cells revealed that canagliflozin potentiated paclitaxel-induced apoptosis and DNA damaging effect. Paclitaxel in the nanomolar range elevated abnormal mitotic cells as well as aneuploid cells, and canagliflozin further enhanced this effect. Furthermore, canagliflozin downregulated cyclin B1 and phospho-BUBR1 upon spindle assembly checkpoint (SAC) activation by paclitaxel, and may consequently impair SAC. Thus, paclitaxel disturbed microtubule dynamics and canagliflozin compromised SAC activity, together they may induce premature mitotic exit, accumulation of aneuploid cells with DNA damage, and ultimately apoptosis.

Anticancer Activity and Molecular Mechanisms of an Ursodeoxycholic Acid Methyl Ester-Dihydroartemisinin Hybrid via a Triazole Linkage in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma is the third most common cause of cancer-related death according to the International Agency for Research on Cancer. Dihydroartemisinin (DHA), an antimalarial drug, has been reported to exhibit anticancer activity but with a short half-life. We synthesized a series of bile acid-dihydroartemisinin hybrids to improve its stability and anticancer activity and demonstrated that an ursodeoxycholic-DHA (UDC-DHA) hybrid was 10-fold more potent than DHA against HepG2 hepatocellular carcinoma cells. The objectives of this study were to evaluate the anticancer activity and investigate the molecular mechanisms of UDCMe-Z-DHA, a hybrid of ursodeoxycholic acid methyl ester and DHA via a triazole linkage. We found that UDCMe-Z-DHA was even more potent than UDC-DHA in HepG2 cells with IC50 of 1 µM. Time course experiments and stability in medium determined by cell viability assay as well as HPLC-MS/MS analysis revealed that UDCMe-Z-DHA was more stable than DHA, which in part accounted for the increased anticancer activity. Mechanistic studies revealed that UDCMe-Z-DHA caused G0/G1 arrest and induced reactive oxygen species (ROS), mitochondrial membrane potential loss and autophagy, which may in turn lead to apoptosis. Compared to DHA, UDCMe-Z-DHA displayed much lower cytotoxicity toward normal cells. Thus, UDCMe-Z-DHA may be a potential drug candidate for hepatocellular carcinoma.

Discovery of New Glucose Uptake Inhibitors as Potential Anticancer Agents by Non-Radioactive Cell-Based Assays.

Tumor cells rely on aerobic glycolysis to support growth and survival, thus require more glucose supply. Glucose transporters GLUTs, primarily GLUT1, are overexpressed in various cancers. Targeting GLUTs has been regarded as a promising anticancer strategy. In this study, we first evaluated 75 potential GLUT1 inhibitors obtained from virtual screening of the NCI chemical library by a high-throughput cell-based method using a fluorescent glucose analogue 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-d-glucose (2-NBDG) in COS-7 and SKOV3 cells that express high levels of GLUT1. Four compounds, #12, #16, #43 and #69, that significantly inhibited glucose uptake were further evaluated using flow cytometry directly measuring 2-NBDG uptake at the single-cell level and a Glucose Uptake-GloTM assay indirectly measuring 2-deoxy-d-glucose uptake in SKOV3, COS-7 or MCF-7 cells. The inhibitory effect on cancer cell growth was also determined in SKOV3 and MCF-7 cells, and #12 exhibited the best growth inhibitory effect equivalent to a known GLUT1 inhibitor WZB117. Although the anticancer effect of the identified potential GLUT1 inhibitors was moderate, they may enhance the activity of other anticancer drugs. Indeed, we found that #12 synergistically enhanced the anticancer activity of metformin in SKOV3 ovarian cancer cells.

The Combination of a Novel GLUT1 Inhibitor and Cisplatin Synergistically Inhibits Breast Cancer Cell Growth By Enhancing the DNA Damaging Effect and Modulating the Akt/mTOR and MAPK Signaling Pathways.

Breast cancer is the most prevalent cancer and the second leading cause of cancer death in women. Cisplatin is a commonly used chemotherapeutic drug for breast cancer treatment. Owing to serious side effects, the combination of cisplatin with other drugs is an effective strategy to simultaneously reduce side effects and increase the anticancer efficacy. GLUT1 is an emerging target for cancer treatment since cancer cells usually consume more glucose, a phenomenon called the Warburg effect. In this study, we found that the combination of cisplatin and a novel GLUT1 inhibitor #43 identified from our previous high-throughput screening exerted a synergistic anticancer effect in MCF-7 and MDA-MB-231 breast cancer cells. Mechanism studies in MCF-7 cells revealed that combination of cisplatin and #43 significantly induced apoptosis, intracellular reactive oxygen species, and loss of mitochondrial membrane potential. Furthermore, #43 enhanced the DNA damaging effect of cisplatin. Akt/mTOR downstream signaling and the ERK signaling pathway usually involved in cell growth and survival were inhibited by the combination treatment. On the other hand, phosphorylation of p38 and JNK, which may be associated with apoptosis, was induced by the combination treatment. Altogether, our data indicate that oxidative stress, DNA damage, the Akt/mTOR and MAPK signaling pathways, and apoptosis may be involved in the synergism of cisplatin and #43 in breast cancer cells.

Nephrotoxic Polypharmacy and Risk of Contrast Medium-Induced Nephropathy in Hospitalized Patients Undergoing Contrast-Enhanced CT.

OBJECTIVE: For unknown reasons, there is discordance among previous reports with regard to the association of contrast medium (CM) with nephropathy and the incidence of nephropathy after contrast-enhanced CT. This study aimed to determine the frequency of and possible factors related to CM-induced nephropathy in hospitalized patients, with an emphasis on detailing coprescriptions with nephrotoxic potential. MATERIALS AND METHODS: Of 1378 inpatients who underwent CT, 208 (15.1%) met the inclusion criteria: receipt of IV iodinated CM and baseline serum creatinine level obtained within 45 days before and within 2 weeks after CT. Patient demographics, clinical characteristics, comorbidity, nephrotoxic comedications (nine classes of drugs), and type of CM administered were retrospectively reviewed. Relationships between CM-induced nephropathy (serum creatinine level increase ≥ 25% or ≥ 0.5 mg/dL after CT) and risk factors were assessed by stepwise multivariate logistic regression. RESULTS: The cohort of 208 subjects had a high number of comorbidities (mean [± SD], 5.8 ± 3.5 diagnoses) and a high rate of receiving nephrotoxic comedications (45.2%). CM-induced nephropathy was detected in 27 (13.0%) patients. Concurrent use of four nephrotoxic agents (odds ratio [OR], 26.250; 95% CI, 3.673-233.993) was the most influential factor associated with CM-induced nephropathy; other predictors included preexisting renal disease (OR, 8.218; 95% CI, 1.622-42.357), baseline serum creatinine level less than 0.7 or greater than or equal to 1.3 mg/dL (OR, 3.463; 95% CI, 1.341-9.025), and hemoglobin level less than 9.3 g/dL (OR, 3.141; 95% CI, 1.087-8.946). CONCLUSION: Among the known risk factors, such as preexisting renal disease, high serum creatinine level, and low hemoglobin level, a statistically significant association was identified between CM-induced nephropathy and concurrent receipt of four nephrotoxic medications. Relevant preventive measures are warranted for individuals at risk, especially hospitalized patients receiving multiple nephrotoxic medications who require contrast-enhanced CT.

Epi-reevesioside F inhibits Na+/K+-ATPase, causing cytosolic acidification, Bak activation and apoptosis in glioblastoma.

Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC50 values of 27.3±1.7 vs. 48.7±1.8 nM (P < 0.001) and 45.0±3.4 vs. 81.3±4.3 nM (P < 0.001) in glioblastoma T98 and U87 cells, respectively. However, both Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na+/K+-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na+ but not Ca2+, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na+/K+-ATPase, leading to overload of intracellular Na+ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.

Calanquinone A induces anti-glioblastoma activity through glutathione-involved DNA damage and AMPK activation.

Glioblastoma, a highly malignant glioma, is resistant to both radiation and chemotherapy and is an intractable problem in clinical treatment. New therapeutic approaches are in urgent need. Calanquinone A, an herbal constituent, displayed anti-proliferative activity against glioblastoma cells, including A172, T98 and U87. Flow cytometric analysis showed an S phase arrest and a subsequent apoptosis to calanquinone A action. Further identification demonstrated a rapid increase of γH2A.X formation at S phase. The data together with comet tail formation and Chk1 activation indicated DNA damage response. N-acetyl cysteine (an antioxidant and a glutathione precursor) and exogenously applied glutathione, but not trolox (an antioxidant), completely abolished calanquinone A-induced effects. Immunofluorescence assay revealed that calanquinone A decreased the intracellular glutathione levels in both A172 and T98 cells. However, calanquinone A, by itself, did not conjugate glutathione. The data suggested that the decrease of cellular glutathione predominantly contributed to the anticancer mechanism. Furthermore, calanquinone A induced the activation of AMP-activated protein kinase (AMPK) and the inhibition of p70S6K activity. Rhodamine efflux assay showed that calanquinone A did not block efflux activity, indicating that calanquinone A was not a P-glycoprotein substrate. In summary, the data suggest that calanquinone A displays anti-glioblastoma activity through a decrease of cellular glutathione levels that subsequently induces DNA damage stress and AMPK activation, leading to cell cycle arrest at S-phase and apoptotic cell death. Furthermore, calanquinone A does not serve as a P-glycoprotein substrate, suggesting a potential for further development in anti-glioblastoma therapy.

Paclitaxel induces apoptosis through activation of nuclear protein kinase C-δ and subsequent activation of Golgi associated Cdk1 in human hormone refractory prostate cancer.

PURPOSE: Emerging evidence shows that the translocation of apoptosis related factors on cellular organelles, such as mitochondria, endoplasmic reticulum, Golgi apparatus and nucleus, has a crucial role in the apoptotic process. We characterized the effect of paclitaxel (Sigma®) on Golgi involved apoptosis in human hormone refractory prostate cancer. MATERIALS AND METHODS: FACScan™ flow cytometric analysis was used to determine cell cycle distribution and the subG1 (apoptosis) population. Protein expression and localization were detected by Western blot, confocal microscopic examination and the sucrose gradient separation technique. RESULTS: Paclitaxel induced Golgi apparatus disassembly and interaction between Golgi complexes and mitochondria. Discontinuous sucrose gradient fractionation was used to determine and collect Golgi containing fractions. Data revealed that paclitaxel induced an increase of Cdk1 activity and DR5 expression on the Golgi complex that was associated with increased cleavage of caspase-8, a DR5 downstream factor, and caspase-3 into catalytically active fragments. Data were validated by confocal immunofluorescence microscopy. Golgi associated effects were inhibited by the Cdk1 inhibitor roscovitine (Sigma), suggesting a critical role for Golgi-Cdk1. Also, paclitaxel caused an increase of nuclear but not of Golgi associated PKC-δ activity. The selective PKC-δ inhibitor rottlerin (Sigma) completely inhibited the increase of Golgi-Cdk1 activity, suggesting that nuclear PKC-δ served as an upstream regulator of Golgi-Cdk1. CONCLUSIONS: Data suggest that paclitaxel induces nuclear translocation and activation of PKC-δ, which in turn causes Golgi-Cdk1 activation, leading to Golgi associated DR5 up-regulation, and caspase-8 and 3 activation. Golgi mediated signaling cascades facilitate mitochondria involved apoptotic pathways and at least partly explain the anticancer activity of paclitaxel action.

Costunolide induces apoptosis through nuclear calcium2+ overload and DNA damage response in human prostate cancer.

PURPOSE: Costunolide is a natural sesquiterpene lactone. We elucidated what to our knowledge is a novel mechanism to highlight its potential in chemotherapy for prostate cancer, particularly androgen refractory prostate cancer. MATERIALS AND METHODS: Several pharmacological and biochemical assays were used to characterize the apoptotic signaling pathways of costunolide (ChromaDex™) in prostate cancer cells. RESULTS: Costunolide showed effective antiproliferative activity against hormone dependent (LNCaP) and independent (PC-3 and DU-145) prostate cancer cells (ATCC®) by sulforhodamine B assay, clonogenic test and flow cytometric analysis of carboxyfluorescein succinimidyl ester labeling. In PC-3 cells data showed that costunolide induced a rapid overload of nuclear Ca(2+), DNA damage response and ATR phosphorylation. Costunolide induced G1-phase cell cycle arrest, which was supported by p21 up-regulation and its association with the cyclin dependent kinase 2/cyclin E complex. The association resulted in inhibition of the complex activity and inhibition of Rb phosphorylation. Costunolide mediated effects were substantially inhibited by glutathione, the reactive oxygen species scavenger and glutathione precursor N-acetylcysteine, and the Ca(2+) chelator BAPTA-AM other than the reactive oxygen species scavenger Trolox®. This indicated the crucial role of intracellular Ca(2+) mobilization and thiol depletion but not of reactive oxygen species production in apoptotic signaling. CONCLUSIONS: Data suggest that costunolide induces the depletion of intracellular thiols and overload of nuclear Ca(2+) that cause DNA damage and p21 up-regulation. The association of p21 with the cyclin dependent kinase 2/cyclin E complex blocks cyclin dependent kinase 2 activity and inhibits Rb phosphorylation, leading to G1 arrest of the cell cycle and subsequent apoptotic cell death in human prostate cancer cells.

Cryptocaryone, a natural dihydrochalcone, induces apoptosis in human androgen independent prostate cancer cells by death receptor clustering in lipid raft and nonraft compartments.

PURPOSE: Androgen refractory prostate cancer is a major clinical challenge. Treatment approaches to prostate cancer are based on various mechanisms that cause malignant cell apoptosis. Of these strategies the anticancer effect of triggering death receptors is well substantiated. MATERIALS AND METHODS: Several pharmacological and biochemical assays were used to characterize the apoptotic signaling pathways of the natural dihydrochalcone cryptocaryone in prostate cancer cells. RESULTS: Cryptocaryone induced antiproliferative and apoptotic effects in human androgen independent prostate cancer cells. It induced caspase-8 and 3 activation but did not change total protein levels of death receptors and their ligands. DR5 surface expression was moderately increased by cryptocaryone. Confocal immunofluorescence examination showed that cryptocaryone induced Fas clustering and the association of downstream signaling molecules, including FADD and procaspase-8. DR4 and DR5 aggregation was also induced by cryptocaryone. Data were confirmed by protein profile analysis of detergent resistant membranes showing that Fas, DR4, DR5, FADD and procaspase-8 levels were increased 1.3, 3.5, 4.1, 13.1 and 4.1-fold, respectively, in the lipid raft compartment. Cryptocaryone mediated clustering of death receptors and associated molecules was also detected in nonraft compartments. The distribution between lipid raft and nonraft compartments was validated by the cholesterol depleting agent methyl-beta-cyclodextrin. Cryptocaryone significantly potentiated FasL induced apoptosis in PC-3 cells. CONCLUSIONS: We suggest that cryptocaryone has anticancer activity via the stimulation of death receptor and associated molecule clustering, leading to caspase-8 and 3 activation, and apoptosis in prostate cancer cells.

Ilimaquinone, a marine sponge metabolite, displays anticancer activity via GADD153-mediated pathway.

The marine organisms produce many metabolic substances with numerous pharmacological activities. It has been suggested that ilimaquinone, a metabolite of sea sponge, can induce vesiculation of the Golgi apparatus and display several biological activities, such as anti-human immunodeficiency virus, anti-inflammation as well as anti-microbial activities. In this study, the sulforhodamine B assays showed that ilimaquinone induced a concentration-dependent anti-proliferative effect in several types of cancer cell lines, including prostate cancer PC-3 and LNCaP, non-small cell lung cancer A549 and hepatocellular carcinoma Hep3B cells. The anticancer mechanism of ilimaquinone in the representative PC-3 cells was identified. Ilimaquinone induced a time-dependent increase of G(1) phase arrest and a subsequent increase of hypodiploid sub-G(1) phase (apoptosis) of the cell cycle. The arrest of the cell cycle was associated with a sustained high level of nuclear cyclin E but the absence of DNA synthesis by flow cytometric analysis, indicating an incomplete S phase. Although ilimaquinone-induced Golgi vesiculation, the data showed that the inhibition of cancer cell growth was not through the Golgi fragmentation. Several biological kinases and transcription factors were examined in this study. The data demonstrated that ilimaquinone did not activate extracellular signal-regulated kinase and phosphatidylinositol 3-kinase but induce the up-regulation and nuclear translocation of growth arrest and DNA damage inducible gene 153 (CHOP/GADD153). Furthermore, ilimaquinone-mediated anti-proliferative effect is significantly reduced in the antisense CHOP/GADD153-overexpressing cells. Ilimaquinone also inhibited DNA binding of NF-kappaB; however, this inhibitory effect could not explain ilimaquinone-induced anticancer effect. In summary, it is suggested that ilimaquinone induces the anti-proliferative effect through the G(1) arrest of the cell cycle and the up-regulation and nuclear translocation of CHOP/GADD153.

Investigation of anti-tumor mechanisms of K2154: characterization of tubulin isotypes, mitotic arrest and apoptotic machinery.

Microtubules are crucial targets for cancer chemotherapeutic drugs, and new microtubule-directed agents are of continued interest in drug development. A novel microtubule-directed agent, ethyl-2-[N-rho-chlorobenzyl-(2'-methoxy)]-anilino-4-oxo -4, 5-dihydro-furan-3-carboxylate, was identified. The compound, designated K2154, inhibited cell proliferation, with IC(50) values of 10.3, 15.3, 9.6, 11.2, 12.8 and 12.1 muM in prostate cancer PC-3, hepatocellular carcinoma Hep3B, non-small cell lung cancer A549, colorectal cancer HT29 and HCT116, and P-glycoprotein-rich breast cancer NCI/ADR-RES cells, respectively. Because NCI/ADR-RES cells were susceptible to inhibition by K2154, it indicated that this compound is a poor substrate for P-glycoprotein. In this study, PC-3 cells were used to identify the anticancer mechanisms of K2154. K2154 induced an arrest of the cell cycle at G2/M phase and a subsequent increase of hypodiploid phase in PC-3 cells, whereas it only induced a moderate level of G2/M arrest with little increase of hypodiploid phase in normal prostate cells. K2154 inhibited microtubule assembly in both in vitro turbidity assay and in vivo microtubule spin-down experiment. Immunochemical examination showed that K2154 caused formation of abnormal mitotic characteristics with bipolar spindles, particularly, in beta(II)- and beta(III)-tubulin staining. It also induced several pathways, including cyclin B1 up-regulation, dephosphorylation on Tyr(15) and phosphorylation on Thr(161) of Cdk1 and Cdc25C phosphorylation, and roscovitine (a Cdk1 inhibitor) significantly inhibited K2154-induced apoptosis, suggesting a pro-apoptotic role of Cdk1. Phosphorylation of Bcl-2 and Bcl-xL and cleavage of Mcl-1, together with activation of caspase-9 and -3, indicated that mitochondrial pathway played a central role in K2154-mediated apoptotic cell death. Additionally, AIF contributed to a late phase of K2154-induced apoptotic pathway. In conclusion, it is suggested that K2154 displays an anticancer activity through a target on microtubules and a subsequent signaling cascade on cell cycle regulation and apoptotic machinery.

Induction of Fas clustering and apoptosis by coral prostanoid in human hormone-resistant prostate cancer cells.

Cyclopentenone prostaglandins (PGs) such as PGA1, PGA2 and delta12-PGJ2 have been shown to suppress tumor cell growth and to induce apoptosis in prostate cancer cells. Bromovulone III, which is isolated from the soft coral Clavularia viridis, is a cyclopentenone prostanoid. In this study, the anti-tumor activity as well as action mechanism of bromovulone III was identified in prostate cancer cells. Bromovulone III displayed anti-tumor activity of 30 to 100 times more effective than PGA1, PGA2 and delta12-PGJ2 in PC-3 cells. Several targets of caspases and Bcl-2 family of proteins were detected and the data demonstrated that bromovulone III induced the activation of caspase-8, -9 and -3, and Bid cleavage in which the caspase-8 activation occurred the first. Bromovulone III did not modify the protein levels of death receptors and ligands. Of note, the Fas clustering in PC-3 cells responsive to bromovulone III was observed by confocal immunofluorescence microscopy suggesting the involvement of Fas-mediated pathway. Bromovulone III also induced the cleavage of Mcl-1 in this study. The cleavage fragments (24, 19 and 17 kDa) may partly share the apoptotic insult. Although it has been suggested that Fas-mediated signaling may contribute to the caspase-8 activation induced by DNA-damaging agents; however, bromovulone III did not induce any DNA breakage, suggesting that bromovulone III-induced Fas/caspase-8-dependent signaling is not through the direct target on DNA damage. In summary, the data suggest that bromovulone III causes a rapid redistribution and clustering of Fas in PC-3 cells. Subsequently, the Fas event causes the activation and interaction of caspase-8/Bid/caspase-9 signaling cascades, and the activation of executor caspase-3.

Investigation of extrinsic and intrinsic apoptosis pathways of new clerodane diterpenoids in human prostate cancer PC-3 cells.

In our continuing search to discover bioactive compounds from natural products, we isolated six new clerodane diterpenes, caseamembrins A to F, from Casearia membranacea and examined their antiproliferative activities in human hormone-resistant prostate cancer PC-3 cells. All of these compounds displayed effective antiproliferative activity using sulforhodamine B assays and induced cell apoptosis by a terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL)-reaction technique. The data demonstrated that caseamembrin C was the most effective compound among these clerodane diterpenoids. Caseamembrin C induced down-regulation of Bcl-2 and Bcl-xL expression, while up-regulation of proapoptotic protein Mcl-1S (short chain), suggesting that these Bcl-2 family member proteins may play a role on arbitrating the apoptotic cell death. Caseamembrin C also induced the up-regulation of Fas ligand (FasL) expression, cleavage and activation of caspase-8 and caspase-9, Bid cleavage and activation of executor caspase-3. However, z-IETD-FMK (Z-Ile-Glu-Thr-Asp-fluoromethyl ketone, a selective caspase-8 inhibitor) almost completely inhibited caseamembrin C-induced Bid cleavage without any modification of caspase-9 activation, indicating that the extrinsic pathway of FasL/caspase-8/Bid cascade only played a minor role in the apoptotic signaling. Taken together, it is suggested that caseamembrin C-induced apoptosis is predominantly through the activation of intrinsic apoptosis pathways by causing the down-regulation of Bcl-2 and Bcl-xL expression, up-regulation of Mcl-1S protein and activation of caspase-9 and caspase-3.