Targeting the ubiquitin-proteasome pathway to overcome anti-cancer drug resistance.

Drug resistance is a major obstacle in the field of pre-clinical and clinical therapeutics. The development of novel technologies and targeted therapies have yielded new modalities to overcome drug resistance, but multidrug resistance (MDR) remains one of the major challenges in the treatment of cancer. The ubiquitin-proteasome system (UPS) has a central role in regulating the levels and activities of a multitude of proteins as well as regulation of cell cycle, gene expression, response to oxidative stress, cell survival, cell proliferation and apoptosis. Therefore, inhibition of the UPS could represent a novel strategy for the treatment and overcoming of drug resistance in chemoresistant malignancies. In 2003, bortezomib was approved by the FDA for the treatment of multiple myeloma (MM). However, due to its limitations, second generation proteasome inhibitors (PIs) like carfilzomib, ixazomib, oprozomib, delanzomib and marizomib were introduced which displayed clinical activity in bortezomib-resistant tumors. Past studies have demonstrated that proteasome inhibition potentiates the anti-cancer efficacy of other chemotherapeutic drugs by: i) decreasing the expression of anti-apoptotic proteins such as TNF-α and NF-kB, ii) increasing the levels of Noxa, a pro-apoptotic protein, iii) activating caspases and inducing apoptosis, iv) degrading the pro-survival protein, induced myeloid leukemia cell differentiation protein (MCL1), and v) inhibiting drug efflux transporters. In addition, the mechanism of action of the immunoproteasome inhibitors, ONX-0914 and LU-102, suggested their therapeutic role in the combination treatment with PIs. In the current review, we discuss various PIs and their underlying mechanisms in surmounting anti-tumor drug resistance when used in combination with conventional chemotherapeutic agents.

The Novel Benzamide Derivative, VKNG-2, Restores the Efficacy of Chemotherapeutic Drugs in Colon Cancer Cell Lines by Inhibiting the ABCG2 Transporter.

The overexpression of ATP-binding cassette transporter, ABCG2, plays an important role in mediating multidrug resistance (MDR) in certain types of cancer cells. ABCG2-mediated MDR can significantly attenuate or abrogate the efficacy of anticancer drugs by increasing their efflux from cancer cells. In this study, we determined the efficacy of the novel benzamide derivative, VKNG-2, to overcome MDR due to the overexpression of the ABCG2 transporter in the colon cancer cell line, S1-M1-80. In vitro, 5 µM of VKNG-2 reversed the resistance of S1-M1-80 cell line to mitoxantrone (70-fold increase in efficacy) or SN-38 (112-fold increase in efficacy). In contrast, in vitro, 5 µM of VKNG-2 did not significantly alter either the expression of ABCG2, AKT, and PI3K p110ß protein or the subcellular localization of the ABCG2 protein compared to colon cancer cells incubated with the vehicle. Molecular docking data indicated that VKNG-2 had a high docking score (-10.2 kcal/mol) for the ABCG2 transporter substrate-drug binding site whereas it had a low affinity on ABCB1 and ABCC1 transporters. Finally, VKNG-2 produced a significant concentration-dependent increase in ATPase activity (EC50 = 2.3 µM). In conclusion, our study suggests that in vitro, VKNG-2 reverses the resistance of S1-M1-80, a cancer cell line resistant to mitoxantrone and SN-38, by inhibiting the efflux function of the ABCG2 transporter.

Synthesis and Cytotoxicity Studies of Stilbene Long-Chain Fatty Acid Conjugates.

A series of 16 conjugates of the tubulin polymerization inhibitor combretastatin A4 (CA-4) and other functionally related stilbene with four 18-carbon fatty acids, namely, stearic, oleic, linoleic, and linolenic acids, have been synthesized in good yields. These new derivatives have been evaluated against the KB-3-1 (human epidermoid carcinoma), NCI-H460 (human lung cancer), HEK293 (human embryonic kidney), and MCF-7 (human breast adenocarcinoma) cell lines for antiproliferative activity, with the exhibited cytotoxic activities comparable with those of CA-4 and colchicine. Compounds 22 and 23, CA-4 conjugates of linoleic and linolenic acids, respectively, were determined to have exhibited the most active in vitro assays, with compound 23 exhibiting very similar activity to the parent compound against the NCI-H460 cell line. Our studies further delineated the structurally required Z-geometry of the stilbene moiety and that conjugation of the less active E-stilbenes with the most active fatty acid had minimal or no improvement in their respective activities.

GSK1904529A, a Potent IGF-IR Inhibitor, Reverses MRP1-Mediated Multidrug Resistance.

Overexpression of multidrug-resistant efflux transporters is one of the major causes of chemotherapy failure. MRP1, a 190 kDa efflux transporter, confers resistance to a wide of range of chemotherapeutic drugs. Here we study the cellular effects of GSK1904529A in reversing MRP1-mediated drug resistance. Cytotoxicity of GSK1904529A was determined by MTT assay. Reversal effects of GSK1904529A in combination with MRP1 substrates were determined. The intracellular accumulation and efflux of MRP1 substrate was measured by scintillation counter and protein expression was determined by Western blotting analysis. Cell cycle effects of GSK1904529A in combination with MRP1 substrates were determined by flow cytometric analysis. GSK1904529A, at non-toxic concentrations, enhanced the cytotoxicity of MRP1 substrates in HEK293/MRP1 cells. Furthermore, GSK1904529A increased the intracellular accumulation of [3 H]-vinblastine by inhibiting the efflux function of MRP1. GSK1904529A did not alter the expression level of MRP1, induced a G0/G1 phase cell cycle arrest. Our results indicated that GSK1904529A significantly increased the sensitivity of MRP1 overexpressing cells to chemotherapeutic agents. Furthermore, GSK1904529A enhanced the efficacy of chemotherapeutic drugs that are substrates of MRP1. J. Cell. Biochem. 118: 3260-3267, 2017. © 2017 Wiley Periodicals, Inc.

ABCB1 limits the cytotoxic activity of TAK-243, an inhibitor of the ubiquitin-activating enzyme UBA1.

BACKGROUND: One of the major concerns of cancer therapy is the emergence of multidrug resistance (MDR). The MDR-associated ATP-binding cassette sub-family B member 1 (ABCB1) transporter is established to mediate resistance against numerous anticancer drugs. In this study, we demonstrated that the Ubiquitin-like modifier activating enzyme 1 (UBA1) inhibitor TAK-243 is transported by the ABCB1. METHODS: MTT assay was performed to evaluate the cytotoxicity of TAK-243. Western blot was carried out to investigate if TAK-243 affect to ABCB1 protein expression in cancer cells. High Performance Liquid Chromatography (HPLC) and ATPase assay were carried out to confirm TAK-243 as an ABCB1 substrate. [3H]-paclitaxel accumulation assay was used to determine the MDR reversal effect of TAK-243. Computational docking analysis was performed to investigate the drug-transporter binding position. RESULTS: The cytotoxicity profile showed that TAK-243 was less effective in ABCB1-overexpressing cells than in the parental cells, but pharmacological inhibition or knockout the gene of ABCB1 was able to reverse TAK-243 resistance. Furthermore, TAK-243 potently stimulated ABCB1 ATPase activity and the HPLC analysis revealed that TAK-243 accumulation was significantly reduced in ABCB1-overexpressing cells. Finally, the computational docking analysis indicates a high binding affinity between TAK-243 and human ABCB1 transporter. CONCLUSIONS: Our in vitro data characterized TAK-243 as a substrate of ABCB1, which may predict limited anticancer effect of this compound in drug resistant tumors.

Anticancer effect of Indanone-based thiazolyl hydrazone derivative on p53 mutant colorectal cancer cell lines: An in vitro and in vivo study.

Colorectal cancer is a major health problem, and it is the third most diagnosed cancer in the United States. The current treatment for colorectal cancer includes irinotecan, a topoisomerase I inhibitor, and other targeted drugs, such as bevacizumab and regorafenib. The low response rates and incidence of high toxicity caused by these drugs instigated an evaluation of the anticancer efficacy of a series of 13 thiazolyl hydrazone derivatives of 1-indanone, and four compounds among them show favorable anticancer activity against some of the tested colorectal cancer cell lines with IC50 values ranging from 0.41 ± 0.19 to 6.85 ± 1.44 µM. It is noteworthy that one of the indanone-based thiazolyl hydrazone (ITH) derivatives, N-Indan-1-ylidene-N'-(4-Biphenyl-4-yl-thiazol-2-yl)-hydrazine (ITH-6), has a better cytotoxicity profile against p53 mutant colorectal cancer cells HT-29, COLO 205, and KM 12 than a p53 wild-type colorectal cancer cell line, such as HCT 116. Mechanistic studies show that ITH-6 arrests these three cancer cell lines in the G2/M phase and induces apoptosis. It also causes a rise in the reactive oxygen species level with a remarkable decrease in the glutathione (GSH) level. Moreover, ITH-6 inhibits the expression of NF-κB p65 and Bcl-2, which proves its cytotoxic action. In addition, ITH-6 significantly decreased tumor size, growth rate, and tumor volume in mice bearing HT-29 and KM 12 tumor xenografts. Moreover, CRISPR/Cas9 was applied to establish an NF-κB p65 gene knockout HT-29 cell line model to validate the target of ITH-6. Overall, the results suggest that ITH-6 could be a potential anticancer drug candidate for p53 mutant colorectal cancers.

Overexpression of ABCG2 Confers Resistance to MLN7243, a Ubiquitin-Activating Enzyme (UAE) Inhibitor.

Overexpression of ATP-binding cassette transporter superfamily G member 2 (ABCG2), is known as a major mechanism mediating multidrug resistance (MDR) in cancer cells. MLN7243 is a small-molecule ubiquitin activating enzyme inhibitor currently under clinical investigation. The aim of the current study is to determine if MLN7243 is a substrate of MDR-related ABCG2 transporter. Our results showed that cancer cells overexpressing ABCG2 transporter were resistant to MLN7243 compared to the parental cells, while knockout of ABCG2 gene or pharmacological inhibition of ABCG2 efflux function completely reversed the drug resistance. Unexpectedly, the endogenous low expression of ABCG2 is sufficient to confer cancer cells resistance to MLN7243. The ABCG2 ATPase assay and HPLC assay suggested that MLN7243 can significantly stimulate ABCG2 ATPase activity and be pumped out from ABCG2-overexpressing cells by ABCG2. The docking analysis also implied that MLN7243 binds to ABCG2 drug-binding pocket with optimal binding affinity. However, MLN7243 did not competitively inhibit the efflux of other ABCG2 substrate drugs, indicating it may not serve as an MDR reversal agent. In conclusion, our study provides direct in vitro evidence to show that MLN7243 is a potent ABCG2 substrate. If our results can be translated to humans, it suggests that combining MLN7243 with ABCG2 inhibitors may enhance the anticancer efficacy for patients with high tumor ABCG2 level.

Cabozantinib Reverses Topotecan Resistance in Human Non-Small Cell Lung Cancer NCI-H460/TPT10 Cell Line and Tumor Xenograft Model.

Cabozantinib (CBZ) is a small molecule tyrosine kinase receptor inhibitor, which could also inhibit the ABCG2 transporter function. Therefore, CBZ could re-sensitize cancer cells that are resistant to ABCG2 substrate drugs including topotecan (TPT). However, its reversal effect against TPT resistance has not been tested in a TPT-induced resistant cancer model. In this study, a new TPT selected human non-small cell lung cancer (NSCLC)-resistant cell model NCI-H460/TPT10 with ABCG2 overexpression and its parental NCI-H460 cells were utilized to investigate the role of CBZ in drug resistance. The in vitro study showed that CBZ, at a non-toxic concentration, could re-sensitize NCI-H460/TPT10 cells to TPT by restoring intracellular TPT accumulation via inhibiting ABCG2 function. In addition, the increased cytotoxicity by co-administration of CBZ and TPT may be contributed by the synergistic effect on downregulating ABCG2 expression in NCI-H460/TPT10 cells. To further verify the applicability of the NCI-H460/TPT10 cell line to test multidrug resistance (MDR) reversal agents in vivo and to evaluate the in vivo efficacy of CBZ on reversing TPT resistance, a tumor xenograft mouse model was established by implanting NCI-H460 and NCI-H460/TPT10 into nude mice. The NCI-H460/TPT10 xenograft tumors treated with the combination of TPT and CBZ dramatically reduced in size compared to tumors treated with TPT or CBZ alone. The TPT-resistant phenotype of NCI-H460/TPT10 cell line and the reversal capability of CBZ in NCI-H460/TPT10 cells could be extended from in vitro cell model to in vivo xenograft model. Collectively, CBZ is considered to be a potential approach in overcoming ABCG2-mediated MDR in NSCLC. The established NCI-H460/TPT10 xenograft model could be a sound clinically relevant resource for future drug screening to eradicate ABCG2-mediated MDR in NSCLC.

VKNG-1 Antagonizes ABCG2-Mediated Multidrug Resistance via p-AKT and Bcl-2 Pathway in Colon Cancer: In Vitro and In Vivo Study.

The emergence of multidrug resistance (MDR) to chemotherapeutic drugs is a major problem in the therapy of cancer. Knowledge of the mechanisms of drug resistance in cancer is necessary for developing efficacious therapies. ATP-binding cassette (ABC) transporters are transmembrane proteins that efflux chemotherapeutic drugs from cancer cells, thereby producing MDR. Our research efforts have led to the discovery of VKNG-1, a compound that selectively inhibits the ABCG2 transporter and reverses resistanctabe to standard anticancer drugs both in vitro and in vivo. VKNG-1, at 6 µM, selectively inhibited ABCG2 transporter and sensitized ABCG2-overexpressing drug-resistant cancer cells to the ABCG2 substrate anticancer drugs mitoxantrone, SN-38, and doxorubicin in ABCG2-overexpressing colon cancers. VKNG- 1 reverses ABCG2-mediated MDR by blocking ABCG2 efflux activity and downregulating ABCG2 expression at the mRNA and protein levels. Moreover, VKNG-1 inhibits the level of phosphorylated protein kinase B (PKB/p-AKT), and B-cell lymphoma-2 (Bcl-2) protein which may overcome resistance to anticancer drugs. However, the in vitro translocation of ABCG2 protein did not occur in the presence of 6 µM of VKNG-1. In addition, VKNG-1 enhanced the anticancer efficacy of irinotecan in ABCG2- overexpressing mouse tumor xenografts. Overall, our results suggest that VKNG-1 may, in combination with certain anticancer drugs, represent a treatment to overcome ABCG2-mediated MDR colon cancers.

The Spleen Tyrosine Kinase Inhibitor, Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance.

Tyrosine kinase inhibitors (TKIs) are important in managing lymphoid malignancies by targeting B-cell receptor signaling pathways. Entospletinib (GS-9973) is an oral, selective inhibitor of spleen tyrosine kinase (Syk), currently in the phase II clinical trials for the treatment of chronic lymphocytic leukemia. Syk is abundantly present in the cells of hematopoietic lineage that mediates cell proliferation, differentiation, and adhesion. In this current study, we evaluated the efficacy of GS-9973 to overcome multidrug resistance (MDR) due to the overexpression of the ABCG2 transporter in the non-small cell lung cancer (NSCLC) cell line, NCI-H460/MX20. In vitro, 3 µM of GS-9973 reversed the drug resistance of NCI-H460/MX20 cell line to mitoxantrone or doxorubicin. GS-9973, at 3 µM reverses ABCG2-mediated MDR by blocking ABCG2 efflux activity and downregulating ABCG2 expression at the protein level but did not alter the ABCG2 mRNA expression and subcellular localization of the ABCG2 protein compared to drug-resistant cells incubated with the vehicle. GS-9973 produced a moderate concentration-dependent increase in the ATPase activity of ABCG2 (EC50 = 0.42 µM) and molecular docking data indicated that GS-9973 had a high affinity (-10.226 kcal/mol) for the substrate-binding site of ABCG2. Finally, HPLC analysis proved that the intracellular concentration of GS-9973 is not significantly different in both parental and resistant cell lines. In conclusion, our study suggests that in vitro, GS-9973 in combination with certain anticancer drugs, represent a strategy to overcome ABCG2-mediated MDR cancers.

Anti-cancer effect of Indanone-based thiazolyl hydrazone derivative on colon cancer cell lines.

Colorectal cancer is the third leading cause of cancer related deaths in the United States. Currently, Irinotecan, a topoisomerase I inhibitor, is an approved anti-cancer drug for the treatment of patients with advanced or recurrent colorectal cancer. Considering low response rate and events of high toxicity caused by irinotecan, we evaluated a series of thirteen thiazolyl hydrazone derivatives of 1-indanone for their potential antineoplastic activity and four compounds showed promising anti-cancer activity against most of the tested colon cancer cell lines with IC50 values ranging from 0.41 ± 0.19 to 6.85 ± 1.44 µM. It is noteworthy that the compound, N-Indan-1-ylidene-N'-(4-Biphenyl-4-yl-thiazol-2-yl)-hydrazine (ITH-6) is found to be more effective than irinotecan against colon cancer cells, HT-29, COLO 205, and KM 12. Mechanistic studies reveal that ITH-6 arrests these cancer cell lines in G2/M phase of the cell cycle, induces apoptosis and causes an increase in ROS level with a significant reduction in the GSH level. The mechanism of inhibition relates to the inhibition of tubulin polymerization in the mitotic phase. These findings suggest that ITH-6 is a novel drug candidate for the treatment of colorectal cancer.

Immuno-oncology agent IPI-549 is a modulator of P-glycoprotein (P-gp, MDR1, ABCB1)-mediated multidrug resistance (MDR) in cancer: In vitro and in vivo.

Phosphoinositide 3-kinase gamma isoform (PI3Kγ) plays a critical role in myeloid-derived cells of the immunosuppressive tumor microenvironment. IPI-549, a recently discovered small molecule selective PI3Kγ inhibitor, is currently under immuno-oncology clinical trials in combination with nivolumab, an anti-PD-1 monoclonal antibody immune checkpoint blocker. The purpose of this study is to investigate whether IPI-549 could reverse P-glycoprotein (P-gp)-mediated MDR when combined with chemotherapeutic substrates of P-gp. Cytotoxicity assays showed that IPI-549 reverses P-gp-mediated MDR in SW620/Ad300 and LLC-PK-MDR1 cells. IPI-549 increases the amount of intracellular paclitaxel and inhibits the efflux of paclitaxel out of SW620/Ad300 cells. ABCB1-ATPase assay showed that IPI-549 stimulates the activity of ABCB1-ATPase. IPI-549 does not alter the expression and does not affect the subcellular localization of P-gp in SW620/Ad300 cells. The combination of IPI-549 with paclitaxel showed that IPI-549 potentiates the anti-tumor effects of paclitaxel in P-gp-overexpressing MDR SW620/Ad300 xenograft tumors. With clinical trials beginning to add newly approved immune checkpoint-based immunotherapy into standard-of-care immunogenic chemotherapy to improve patient outcomes, our findings support the rationale of adding IPI-549 to both the chemotherapeutic and immunotherapeutic aspects of cancer combination treatment strategies.

Assessment of cell viability and permeation enhancement in presence of lipid-based self-emulsifying drug delivery systems using Caco-2 cell model: Polysorbate 80 as the surfactant.

PURPOSE: Lipid-based self-emulsifying drug delivery systems (SEDDS) are commonly used for solubilizing and enhancing oral bioavailability of poorly water-soluble drugs. However, their effects on viability of intestine epithelial cells and influence on membrane permeation are poorly understood. The present study was undertaken for safety assessment of lipid-based formulations containing medium-chain fatty acid esters as lipids and polysorbate 80 as the surfactant using the Caco-2 in vitro model. Any possible paracellular permeation enhancement through Caco-2 monolayers by the nontoxic formulations was also investigated. METHODS: Mixtures of monoglyceride (Capmul MCM EP or 708G) or propylene glycol monoester (Capmul PG-8 NF) of medium chain fatty acids with polysorbate 80, with and without the incorporation of a medium-chain triglyceride (Captex 355), were prepared. After suitable dilution with aqueous culture medium, the formulations were incubated with a series of Caco-2 cultures of different maturity. Cell viability and membrane integrity were assessed. Any effects of nontoxic formulations on the transport of the fluorescent dye, Lucifer yellow, through Caco-2 monolayers were also determined. RESULTS: Formulations containing 1:1 ratios of monoglyceride or propylene glycol monoester to triglyceride (30% polysorbate 80, 35% monoglyceride or monoester and 35% triglyceride) were best tolerated by Caco-2 cells. Increased maturity obtained through longer culture durations rendered Caco-2 cells greater tolerance towards lipid-based formulations, and maximum tolerance to lipid-based formulations was observed with Caco-2 monolayers after being cultured for 21-23days. Furthermore, extent of cell membrane rupture caused by lipid-surfactant mixtures correlated positively with levels of cytotoxicity, suggesting a potential underlying mechanism. Permeation studies using Caco-2 monolayer model revealed that certain formulations significantly enhanced paracellular transport activities. CONCLUSIONS: Lipid-based SEDDS containing mixtures of monoglyceride (or monoester) and triglyceride of medium chain fatty acids formed fine microemulsions and were significantly less toxic than other formulations. Fully differentiated Caco-2 monolayer was more resistant to lipid-surfactant mixtures than less mature cultures. Certain formulations were also capable of enhancing paracellular permeation.

Cytotoxicity assessment of lipid-based self-emulsifying drug delivery system with Caco-2 cell model: Cremophor EL as the surfactant.

PURPOSE: Caco-2 cells are used extensively for in vitro prediction of intestinal drug absorption. However, toxicity of excipients and formulations used can artificially increase drug permeation by damaging cell monolayers, thus providing misleading results. The present study aimed to investigate cytotoxicity of common lipid-based excipients and formulations on Caco-2 cells. METHODS: Medium-chain monoglycerides alone or in mixture with the surfactant Cremophor EL, with and without a medium-chain triglyceride, were prepared and incubated with Caco-2 cells from a series of culture stages with varying maturity. Cell viability was evaluated and cell membrane integrity assessed. RESULTS: Cytotoxicity of lipid-based formulations was influenced by the maturity of Caco-2 cells and formulation composition. One-day culture was most sensitive to lipids. When cultured for 5days, viability of Caco-2 cells was significantly improved. The 21-day Caco-2 monolayers maintained the highest survival rate. Microemulsion formulations exhibited significantly less cytotoxicity than neat lipids or surfactant at all stages of cell maturity, and microemulsions containing 1:1 mixtures of monoglyceride and triglyceride appeared to be best tolerated among all the formulations tested. Mechanistically, the observed cytotoxicity was partially due to lipid-induced rupture of cell membrane. CONCLUSIONS: Microemulsions of lipid-surfactant mixtures have less cytotoxicity than lipid alone. Maturity of Caco-2 cells renders significant resistance to cytotoxicity, and monolayers with 21-day maturity are more relevant to in vivo conditions and appear to be a more accurate in vitro model for cytotoxicity assessment.