Amit K. Tiwari

PD173074, a selective FGFR inhibitor, reverses ABCB1-mediated drug resistance in cancer cells.

PurposeSpecific tyrosine kinase inhibitors were recently reported to modulate the activity of ABC transporters, leading to an increase in the intracellular concentration of their substrate drugs. In this study, we determine whether PD173074, a specific fibroblast growth factor receptor (FGFR) inhibitor, could reverse ABC transporter-mediated multidrug resistance.Methods3-(4,5-Dimethylthiazol-yl)-2,5-diphenyllapatinibrazolium bromide assay was used to determine the effect of PD173074 on reversal of ABC transporter-mediated multidrug resistance (MDR). In addition, [3H]-paclitaxel accumulation/efflux assay, western blotting analysis, ATPase, and photoaffinity labeling assays were done to study the interaction of PD173074 on ABC transporters.ResultsPD173074 significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to substrate anticancer drugs colchicine, paclitaxel, and vincristine. This effect of PD173074 is specific to ABCB1, as no significant interaction was detected with other ABC transporters such as ABCC1 and ABCG2. The observed reversal effect seems to be primarily due to the decreased active efflux of [3H]-paclitaxel in ABCB1 overexpressing cells observed in efflux assay. In addition, no significant change in the ABCB1 expression was observed when ABCB1 overexpressing cells were exposed to 5xa0μM PD173074 for up to 3xa0days, thereby further suggesting its role in modulating the function of the transporter. In addition, PD173074 stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Interestingly, PD173074 did not inhibit photolabeling of ABCB1 with [125I]-iodoarylazidoprazosin (IAAP), showing that it binds at a site different from that of IAAP in the drug-binding pocket.ConclusionsHere, we report for the first time, PD173074, an inhibitor of the FGFR, to selectively reverse ABCB1 transporter-mediated MDR by directly blocking the efflux function of the transporter.

IND-2, a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline derivative, circumvents multi-drug resistance and causes apoptosis in colon cancer cells.

Naturally occurring condensed quinolines have anticancer properties. In efforts to find active analogues, we designed and synthesized eight polycyclic heterocycles with a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline framework (IND series). The compounds were evaluated for activity against colon (HCT-116 and S1-MI-80), prostate (PC3 and DU-145), breast (MCF-7 and MDAMB-231), ovarian (ov2008 and A2780), and hepatocellular (HepG2) cancer cells and against non-cancerous Madin Darby canine kidney (MDCK), mouse embryonic fibroblast (NIH/3T3), and human embryonic kidney cells (HEK293). IND-2, a 4-chloro-2-methyl pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline, exhibited more than ten-fold selectivity and potent cytotoxic activity against colon cancer cells relative to the other cancer and non-cancer cells. With five additional colon cancer cell lines (HT-29, HCT-15, LS-180, LS-174, and LoVo), IND-2 had similar cytotoxicity and selectivity, and sub-micromolar concentrations caused changes in the morphology of HCT-116 and HCT-15 cells. IND-2 did not activate the transactivating function of the pregnane X receptor (PXR), indicating that it does not induce PXR-regulated ABCB1 or ABCG2 transporters. Indeed, IND-2 was not a substrate of ABCB1 or ABCG2, and it induced cytotoxicity in HEK293 cells overexpressing ABCB1 or ABCG2 to the same extent as in normal HEK293 cells. IND-2 was cytotoxic to resistant colon carcinoma S1-MI-80 cells, approximately three- and five-fold more than SN-38 and topotecan, respectively. In HCT-116 colon cancer cells, IND-2 produced concentration-dependent changes in mitochondrial membrane potential, leading to apoptosis, and sub-micromolar concentrations caused chromosomal DNA fragmentation. These findings suggest that, by increasing apoptosis, IND-2 has potential therapeutic efficacy for colorectal cancer.

Design, synthesis and in vitro cell-based evaluation of the anti-cancer activities of hispolon analogs

Phytochemicals play an important role in cancer therapy. Hispolon and 26 of its analogs (9 known and 17 new) were synthesized and evaluated for their antiproliferative activities in a panel of six independent human cancer cell lines using the in vitro cell-based MTT assay. Among the hispolon analogs tested, compound VA-2, the most potent overall, produced its most significant effect in the colon cancer cell lines HCT-116 (IC₅₀ 1.4 ± 1.3 μM) and S1 (IC₅₀ 1.8 ± 0.9 μM) compared to its activity in the normal HEK293/pcDNA3.1 cell line (IC₅₀ 15.8±3.7 μM; p<0.01 for each comparison). Based on our results, VA-2 was about 9- to 11-times more potent in colon cancer cells and 2- to 3-times more potent in prostate cancer cells compared to HEK293/pcDNA3.1 cells. Morphological analysis of VA-2 showed significant reduction of cell number, while the cells sizes were also markedly increased and were obvious at 68 h of treatment with 1 μM in HCT-116 (colon) and PC-3 (prostate) cancer cells. A known analog, compound VA-4, prepared by simple modifications on the aromatic functional groups of hispolon, inhibited prostate and colon cancer cell lines with IC₅₀ values <10 μM. In addition, hispolon isoxazole and pyrazole analogs, VA-7 and VA-15 (known), respectively, have shown significant activity with the mean ICv values in the range 3.3-10.7 μM in all the cancer cell lines tested. Activity varied among the analogs in which aromatic functional groups and β-diketone functional groups are modified. But the activity of analogs VA-16 to VA-27 was completely lost when the side chain double-bond was hydrogenated indicating the crucial role of this functionality for anticancer activity. Furthermore, many of the compounds synthesized were not substrates for the ABCB1-transporter, the most common cause of multidrug resistance in anti-cancer drugs, suggesting they may be more effective anticancer agents.

Repurposing phosphodiesterase-5 inhibitors as chemoadjuvants.

Phosphodiesterase-5 (PDE5) inhibitors have shown a beneficial effect in a variety of clinical conditions, such as benign prostate hyperplasia, pulmonary arterial hypertension, female sexual arousal disorder, overactive bladder, and incontinence, Raynauds disease, heart failure and stroke among others (Sandner et al., 2007). Three PDE5 inhibitors, sildenafil (Viagra™), tadalafil (Cialis™) and vardenafil (Levitra™) are clinically approved and are widely used for the treatment of erectile dysfunction. A retrospective analysis determined that men treated with PDE5 for ED had less chance of having prostate cancer. This population of men had significantly lower documented diagnosis of elevated prostate-specific antigen and higher percentage of benign prostatic hyperplasia compared to men not treated with PDE-5 inhibitors (Chavez et al., 2013). Emerging evidence indicates that PDE5 inhibitors are multi-targeting agents and have promising results in the treatment of variety of tumors. Here we propose the possibility of repurposing of PDE5 inhibitors for adjuvant chemotherapy. n nPDE5 contributes to the regulation of intracellular cyclic GMP (cGMP) pools (see Figure u200bFigure1)1) that have been shown to be decreased along with protein kinase-G (PKG), a downstream effector of cGMP, in variety of different tumors such as breast cancer, colon cancer and human oral squamous cell carcinoma (hOSCC) (Spoto et al., 2003; Zhu and Strada, 2007; Di et al., 2010). PDE5 hydrolyzes the 3′, 5′-phosphodiester bond in the second messenger molecule cGMP to biologically inactive 5′-GMP. There is an incomplete understanding of how PDE5 inhibitors act in cancer, yet there are reports of increased apoptosis in different tumor cell types following treatment with PDE5 inhibitors. Possible mechanisms of these anticancer effects via PDE5 inhibition mediated caspase-dependent apoptosis and cell growth arrest may be linked to concomitant increases in regulation of downstream pathways through increased cGMP-PKG levels and subsequent effects on, (1) activation of c-Jun NH2-terminal kinase (JNK), especially JNK1 pathways via phosphorylation of mitogen-activated protein kinase kinase kinase 1 (MEKK1) (Bender and Beavo, 2006), (2) decreased Wnt/β-catenin expression and down-regulation of cyclin D1 (Thompson et al., 2000; Li et al., 2002; Tinsley et al., 2011), (3) inhibition of extracellular-signal regulated kinases 1/2 (ERK1/2) and alterations in the regulation of p42/p44 mitogen activated-protein kinase (MAPK) and p21 pathways (Hou et al., 2006; Das et al., 2008). Increased PDE5 expression was shown to play a role in tumorigenesis in variety of cancers, such as non-small cell lung cancer, urinary bladder cancer, metastatic breast cancer and development of hOSCC (Piazza et al., 2001; Pusztai et al., 2003; Whitehead et al., 2003). Thus, it is hypothesized that inhibiting PDE5 activity may produce antineoplastic actions. There is a small amount of supporting data. Indeed, PDE5 inhibitors, sildenafil and vardenafil induced caspase-dependent apoptosis of B-cell chronic lymphocytic leukemia cells (Sarfati et al., 2003), whereas cytotoxic and growth suppressive effects in various breast cancer, prostate, and colon cells were seen with non-specific PDE5 inhibitors sulindac sulfone and its analogs (Thompson et al., 2000; Piazza et al., 2001; Whitehead et al., 2003; Tinsley et al., 2011). Interestingly, PDE5 inhibitors were shown to alter the tumor microenvironment by augmenting endogenous antitumor immunity by reducing myeloid-derived suppressor cell function (Serafini et al., 2006). n n n nFigure 1 n nSchematic model of PDE5 inhibitors mechanism as chemoadjuvants: Proposed model is shown (A) as how tumor cells can efflux cGMP and variety of anticancer drugs that are substrate of ABCB1, ABCG2, ABCC4, ABCC5, and ABCC10 and so survive in the absence of ... n n n nThe accumulating body of evidence suggests that PDE5 inhibitors could interfere with the efflux functions of the ABC transporters, thus sensitizing cancer cells toward cytotoxic agents that are substrates of ABC transporters (Ding et al., 2011; Shi et al., 2011; Chen et al., 2012) (see Figure u200bFigure1).1). For example, cGMP is a substrate of ABCC4 and ABCC5 transporters that are involved in reducing its intracellular concentrations. Sildenafil reverses this phenomenon through a dual action, first it inhibits PDE5, and secondly it could block the transport function of ABCC4 and ABCC5, thus increasing the intracellular cGMP concentrations (Jedlitschky et al., 2000; Chen et al., 2001). More recently our group reported that specific PDE5 inhibitors could block the function of ABC transporters at clinically achievable concentrations (Ding et al., 2011; Shi et al., 2011; Chen et al., 2012). We showed that sildenafil could block the efflux functions of ABCB1 and ABCG2 transporters in cancer cells, and thus, significantly reversed the MDR-mediated efflux of substrate anticancer drugs, such as mitoxantrone, paclitaxel, and vinca alkaloids (Shi et al., 2011). Other PDE5 inhibitors, vardenafil and tadalafil were also examined for their effect on ABC transporter-mediated efflux in cancer cells. It was found that vardenafil in a concentration-dependent manner, significantly potentiated the cytotoxicity of anticancer agents that are substrates of ABCB1, but not that of ABCC1 or ABCG2 transporters and this effect was significantly greater than that of tadalafil (Ding et al., 2011). Furthermore, sildenafil and vardenafil enhanced the activity of paclitaxel, docetaxel and vinblastine in the ABCC10-transfected HEK293 cells (Chen et al., 2012). Recently, we found that sildenafil significantly enhanced the sensitivity of specific anticancer drugs in different tumor cell lines and in ABCB1- and ABCG2-bearing MDR mouse models (unpublished data). These novel functions of PDE5 inhibitors might explain why in brain tumor models, doxorubicin and herceptin transport efficacy across the blood-brain tumor barrier was enhanced by the addition of sildenafil and vardenafil (Black et al., 2008). Combination chemotherapy with PDE5 inhibitors was shown to produce reactive oxygen species and led to apoptosis that proved to be beneficial in treatment of broad range of cancers. For example, enhanced tumor suppression and apoptotic activity was seen with a sulindac-docetaxel combination in non-small cell lung cancer orthotopic lung tumor model (Whitehead et al., 2003), and with a sulindac-capecitabine combination in breast cancer (Pusztai et al., 2003), and more recently, with the combination of sildenafil-doxorubicin in in vivo models of prostate cancer (Das et al., 2010). We suspect that these combination therapies may have produced enhanced anticancer activity partly due to inhibition of specific ABC transporters, which otherwise reduced intracellular concentration of substrate anticancer agents, but this hypothesis needs to be tested. Furthermore, since the PDE5 inhibitors are substrates of multiple ABC transporters, their individual or overlapping roles (Tiwari et al., 2013) in PDE5 disposition is an open area of research. n nIn summary, there is evidence that suggests that PDE5 inhibitors may have an anticancer action either by increasing cGMP-PKG and coupled downstream events or by their ability to inhibit ABC transporter—mediated s efflux of anticancer drugs. The safety, high tolerability, and wide availability of PDE5 inhibitors have made this class of drug an attractive tool to investigate their role in cancer chemotherapy. Since a detailed understanding of PDE5 inhibitors as anticancer adjuvants is limited, further studies are warranted to characterize their mechanisms and establish their role.

l H -Pyrazolo[3,4- b ]quinolin-3-amine derivatives inhibit growth of colon cancer cells via apoptosis and sub G1 cell cycle arrest

A series of lH-pyrazolo[3,4-b]quinolin-3-amine derivatives were synthesized and evaluated for anticancer efficacy in a panel of ten cancer cell lines, including breast (MDAMB-231 and MCF-7), colon (HCT-116, HCT-15, HT-29 and LOVO), prostate (DU-145 and PC3), brain (LN-229), ovarian (A2780), and human embryonic kidney (HEK293) cells, a non-cancerous cell line. Among the eight derivatives screened, compound QTZ05 had the most potent and selective antitumor efficacy in the four colon cancer cell lines, with IC50 values ranging from 2.3 to 10.2u202fµM. Furthermore, QTZ05 inhibited colony formation in HCT-116 cells in a concentration-dependent manner. Cell cycle analysis data indicated that QTZ05 caused an arrest in the sub G1 cell cycle in HCT-116 cells. QTZ05 induced apoptosis in HCT-116 cells in a concentration-dependent manner that was characterized by chromatin condensation and increase in the fluorescence of fluorochrome-conjugated Annexin V. The findings from our study suggest that QTZ05 may be a valuable prototype for the development of chemotherapeutics targeting apoptotic pathways in colorectal cancer cells.

Abstract B57: Gnetin C, a novel resveratrol dimer, targets pancreatic cancer metabolism

Background: Pancreatic ductal adenocarcinoma (PDAC) is aggressively invasive and treatment-resistant malignancy, and is the fourth leading cause of cancer deaths in the United States. The pancreatic cancer cells alter specific metabolic pathways to meet their tremendous energy and biomass demands which contribute to the progression and dissemination of this disease. More importantly, the oncogenic signaling enables cancer cells to reprogram the cellular metabolism that afford growth and proliferative advantages over normal cells and, thus, may contribute to pancreatic cancer pathophysiology. There is an increasing interest to investigate the oncogenic signaling that controls the metabolic reprogramming of cancer cells. Objectives: Mammalian Target of Rapamycin (mTOR), a central downstream target of Akt, is a highly conserved protein kinase, and is frequent activated in PDAs. Earlier studies reported that mTOR, a master regulator of cell growth and proliferation downstream of oncogenic signaling pathways, controls specific aspects of cellular metabolism through the induction of metabolic gene expression. In our earlier microarray studies, we observed differential expression of genes associated with glucose and lipid metabolic pathways, with concomitant increase in mTOR expression. However, the mechanism and critical link between mTOR and the pancreatic cancer metabolism has not been fully understood. Methodology: In the present study, we evaluated the effectiveness of Melinjo (Indonesian name; Gnetum gnemon L.) seed extract (MSE) and gnetin C (GC), a resveratrol dimer found abundantly in MSE in human (Aspc-1 and PANC-1) and mouse (Pan-02) pancreatic cancer cells. MSE has been reported to exert antioxidant, lipase and amylase inhibition, anti-metabolic syndrome effects and anticancer activities. A recent clinical study reported that MSE decreased serum uric acid and increased HDL cholesterol levels in humans, suggesting that MSE may improve lipid metabolism. To test the anti-tumor activities, pancreatic cancer cells were treated with various concentrations of MSE (0 - 400μg/ml) and GC (0 – 100μM) for 48 h. MTS cell proliferation and apoptotic assays were performed to determine the cell growth inhibition and induction of apoptosis. Trans-resveratrol (RES) that is abundant in grapes and wine was used to compare the effects of GC. Real-time PCR was performed to evaluate the effect of a MSE or GC on mTOR and its downstream targets, as well selected glucose and lipid metabolic pathway genes. Western blot was performed to confirm their expression at the protein level. Results and conclusion: Human (ASPC-1 and PANC-1), and mouse (Pan-02) pancreatic cancer cells treated with MSE, GC and RES showed a dose-dependent decrease in cancer cell proliferation. The IC50 values of MSE, GC and RES related to their anti-proliferative effects revealed that GC, the major metabolite of MSE has superior anti-proliferative properties than resveratrol. Further, flow cytometry analysis showed a pre-G1 peak of apoptosis (>10%) with GC. We observed that GC significantly downregulated mTOR complex, namely (mTORC1 and mTOC2). mTORC1 is a sensor of systemic and local levels of nutrients that regulates cancer cell proliferation and survival. mTORC2 is associated with the ribosome and insulin-stimulated oncogenic PI3K signaling. We also found that GC downregulated p-ribosomal protein S6 (p-RPS6), a downstream target of mTOR that plays important role in both neoplastic and inflammatory processes. In addition, GC also downregulated glycogen synthase kinase 3 alpha and beta (GSK3α and β) that is linked to glycogen metabolism and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), which is associated with lipids and lipoproteins metabolism (cholesterol biosynthesis). Further analysis on key genes of the lipid metabolism is in progress. Given the preliminary evidence that GC has superior anti-proliferative activities and its ability to inhibit mTOR and metabolic/lipid pathway genes can be expected to yield more potent therapies against this deadly disease. The future goal will be to test the potentials of GC on the antitumor efficacy and the effect on metabolic pathway targets in preclinical animal models of pancreatic cancer. Citation Format: Narayanan K. Narayanan, Kazuhiro Kunimasa, Di Tian, Lori Horton, Igor Dolgaev, Adriana Heguy, George Miller, Amit Tiwari, Bhagavathi A. Narayanan. Gnetin C, a novel resveratrol dimer, targets pancreatic cancer metabolism. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B57.