Loperamide potentiates doxorubicin sensitivity in triple-negative breast cancer cells by targeting MDR1 and JNK and suppressing mTOR and Bcl-2: In vitro and molecular docking study.

Multidrug resistance (MDR) is the leading cause of treatment failure in triple-negative breast cancer (TNBC) patients treated with doxorubicin (DXR). We aimed to investigate the potential of the antidiarrheal drug Loperamide (LPR) in sensitizing TNBC cells to DXR and elucidate the underlying molecular mechanisms. Therefore, we examined the effects of DXR alone or in combination with LPR on MDA-MD-231 cells viability using MTT assay, cell cycle, and apoptosis by flow cytometry, and the expression of the MDR-related genes (MDR1 and JNK1) and cell cycle/survival genes (p21, mTOR, and Bcl-2) by quantitative reverse transcription polymerase chain reaction. Results showed that adding LPR to DXR potentiated its antiproliferation effect and reduced its IC50 by twofolds compared with DXR alone. The value of the combination index of LPR/DXR was <1 indicating a synergistic effect. Combined DXR/LPR treatment also caused G1 arrest and potentiated apoptosis more than DXR-single treatment. At the molecular levels, LPR/DXR treatment downregulated the mRNA of MDR1 (1.35-folds), JNK1 (2.5-folds), mTOR (6.6-folds), Bcl-2 (9.5-folds); while upregulated p21 gene (8-folds) compared with DXR alone. Molecular docking analyses found LPR antagonizes MDR1 and JNK1 proteins, and hence supports the in vitro studies. In conclusion, the results confirmed the potential of LPR in sensitizing TNBCs to DXR by targeting MDR1 and JNK1 and suppressing Bcl-2 and mTOR genes, while upregulating the cell cycle inhibitor gene p21. Additionally, LPR could be repurposed to reduce the therapeutic doses of DXR as indicated by the dose reduction index (DRI) and subsequently decrease its side effects.

Antimicrobial Peptides as New Combination Agents in Cancer Therapeutics: A Promising Protocol against HT-29 Tumoral Spheroids.

Antimicrobial peptides are molecules synthetized by a large variety of organisms as an innate defense against pathogens. These natural compounds have been identified as promising alternatives to widely used molecules to treat infections and cancer cells. Antimicrobial peptides could be viewed as future chemotherapeutic alternatives, having the advantage of low propensity to drug resistance. In this study, we evaluated the efficiency of the antimicrobial peptide gramicidin A (GA) and the anticancer drug, doxorubicin (Doxo) against the spheroids from colorectal cancer cells (HT-29). The two drugs were applied separately against HT-29 spheroids as well as together to determine if they can act synergistically. The spheroid evolution, cell viability, and ATP levels were monitored at 24 and 48 h after the applied treatments. The results show significant drops in cell viability and cellular ATP levels for all the experimental treatments. The simultaneous use of the two compounds (GA and Doxo) seems to cause a synergistic effect against the spheroids.

Ribavirin augments doxorubicin's efficacy in human hepatocellular carcinoma through inhibiting doxorubicin-induced eIF4E activation.

Activation of eukaryotic translation initiation factor 4E (eIF4E) is a cellular survival mechanism in response to chemotherapy in cancers. In this work, we demonstrate that targeting eIF4E by ribavirin sensitizes hepatocellular carcinoma (HCC) cell response to doxorubicin. Ribavirin inhibits growth and survival of HCC cells, and to a greater extent than in normal liver cells. Its combination with doxorubicin achieves greater efficacy than single drug in vitro and in vivo. Ribavirin suppresses phosphorylation of molecules involved in Akt/mTOR/eIF4E pathway. Overexpression of the phosphomimetic form (S209D) but not the nonphosphorylatable form (S209A) eIF4E significantly reverses the inhibitory effects of ribavirin. Interestingly, doxorubicin significantly increases p-eIF4E(S209) level in a dose- and time-dependent manner, suggesting that doxorubicin induces eIF4E activation in HCC cells. In addition, eIF4E activation induced by doxorubicin in HCC cells is inhibited by ribavirin. Our work demonstrates the greater efficacy of ribavirin and doxorubicin combination and its underlying mechanisms.

mPEGylated solanesol micelles as redox-responsive nanocarriers with synergistic anticancer effect.

We prepared an amphiphilic redox-responsive conjugate based on mPEGylated solanesol, solanesyl poly(ethylene glycol) dithiodipropionate (SPDP), along with its inert counterpart solanesyl poly(ethylene glycol) succinate (SPGS), which self-assembled in aqueous solution to form redox-responsive micelles. Used as efficient drug carriers for doxorubicin (DOX), the micelles acted as synergistic agents for cancer therapy. Dynamic light scattering (DLS) measurements showed that the SPDP micelles had average diameters of 111nm, which decreased to 88nm after the encapsulation of DOX. The mean diameters and size distribution of the disulfide-containing micelles changed obviously in the presence of the reducing agent glutathione (GSH), whereas no changes occurred in the case of redox-insensitive SPGS micelles. DOX could be loaded into both types of micelles, with drug loading content of about 4.0%. A significantly accelerated release of DOX was triggered by GSH for DOX-loaded SPDP micelles, compared with DOX-loaded SPGS micelles. Blank SPGS and SPDP micelles displayed higher inhibition of HeLa and MCF-7 cell proliferation but less cytotoxicity to normal L-02 cells at similar concentrations. Confocal microscopic observation indicated that a greater amount of DOX was delivered into the nuclei of cells following 9 or 12h incubation with DOX-loaded micelles. In vivo studies on H22-bearing Swiss mice demonstrated the superior anticancer activity of DOX-loaded SPDP micelles over free DOX and DOX-loaded SPGS micelles. All of the data presented here suggested that these SPDP micelles may have a dual function, as they are preferentially toxic for tumor cells alone and are efficient and safe carriers for anticancer drugs. STATEMENT OF SIGNIFICANCE: Various nanoscale drug carriers were used to enhance therapeutic effect of many drugs. While, the metabolites of high quantities of carriers may cause additional short- or long-term toxicities. In this study, a new systems based on solanesol derivatives was developed for anticancer drug delivery. There are two features for this system. One is solanesol originated bioactivity of the carrier, which will synergistically facilitate therapeutic effect of the encapsulated drug. The other is the redox-responsive drug release behavior adaptable to the glutathione-rich atmosphere of tumor cell. All the hypothesis have been elucidated in this work through in vitro and in vivo studies. It was found that this drug delivery system may have a dual function, as they are preferentially toxic for tumor cells alone and are efficient and safe carriers for anticancer drugs.

Chemical Activation in Blood Serum and Human Cell Culture: Improved Ruthenium Complex for Catalytic Uncaging of Alloc-Protected Amines.

Chemical (as opposed to light-induced) activation of caged molecules is a rapidly advancing approach to trigger biological processes. We previously introduced the ruthenium-catalyzed release of allyloxycarbonyl (alloc)-protected amines in human cells. A restriction of this and all other methods is the limited lifetime of the catalyst, thus hampering meaningful applications. In this study, we addressed this problem with the development of a new generation of ruthenium complexes for the uncaging of alloc-protected amines with superior catalytic activity. Under biologically relevant conditions, we achieved a turnover number >300, a reaction rate of 580 m-1 s-1 , and we observed high activity in blood serum. Furthermore, alloc-protected doxorubicin, as an anticancer prodrug, could be activated in human cell culture and induced apoptosis with a single low dose (1 µm) of the new catalyst.

Redox-responsive mesoporous selenium delivery of doxorubicin targets MCF-7 cells and synergistically enhances its anti-tumor activity.

To reduce the side effects and enhance the anti-tumor activities of anticancer drugs in the clinic, the use of nano mesoporous materials, with mesoporous silica (MSN) being the best-studied, has become an effective method of drug delivery. In this study, we successfully synthesized mesoporous selenium (MSe) nanoparticles and first introduced them to the field of drug delivery. Loading MSe with doxorubicin (DOX) is mainly driven by the physical adsorption mechanism of the mesopores, and our results demonstrated that MSe could synergistically enhance the antitumor activity of DOX. Coating the surface of MSe@DOX with Human serum albumin (HSA) generated a unique redox-responsive nanoparticle (HSA-MSe@DOX) that demonstrated glutathione-dependent drug release, increased tumor-targeting effects and enhanced cellular uptake throug nanoparticle interact with SPARC in MCF-7 cells. In vitro, HSA-MSe@DOX prominently induced cancer cell toxicity by synergistically enhancing the effects of MSe and DOX. Moreover, HSA-MSe@DOX possessed tumor-targeting abilities in tumor-bearing nude mice and not only decreased the side effects associated with DOX, but also enhanced its antitumor activity. Therefore, HSA-MSe@DOX is a promising new drug that warrants further evaluation in the treatments of tumors. STATEMENT OF SIGNIFICANCE: To reduce the side effects and enhance the anti-tumor activities of anticancer drugs, we successfully synthesized mesoporous selenium (MSe) nanoparticles and first introduced them to the field of drug delivery. Loading MSe with doxorubicin (DOX) is mainly driven by the physical adsorption mechanism of the mesopores. Coating the surface of MSe@DOX with Human serum albumin (HSA) generated a unique redox-responsive nanoparticle (HSA-MSe@DOX) that demonstrated glutathione-dependent drug release, increased tumor-targeting effects and enhanced cellular uptake throug nanoparticle interact with SPARC in MCF-7 cells. In vitro and in vivo, HSA-MSe@DOX possessed tumor-targeting abilities and not only decreased the side effects associated with DOX, but also enhanced its antitumor activity. Therefore, HSA-MSe@DOX is a promising new drug that warrants further evaluation in the treatments of tumors.

Chemical screening identifies the ß-Carboline alkaloid harmine to be synergistically lethal with doxorubicin.

Despite being an invaluable chemotherapeutic agent for several types of cancer, the clinical utility of doxorubicin is hampered by its age-related and dose-dependent cardiotoxicity. Co-administration of dexrazoxane as a cardioprotective agent has been proposed, however recent studies suggest that it attenuates doxorubicin-induced antitumor activity. Since compounds of natural origin present a rich territory for drug discovery, we set out to identify putative natural compounds with the view to mitigate or minimize doxorubicin cardiotoxicity. We identify the DYRK1A kinase inhibitor harmine, which phosphorylates Tau that is deregulated in Alzheimer's disease, as a potentiator of cell death induced by non-toxic doses of doxorubicin. These observations suggest that harmine or other compounds that target the DYRK1A kinase my offer a new therapeutic opportunity to suppress doxorubicin age-related and dose-dependent cardiotoxicity.

Melissa officinalis Protects against Doxorubicin-Induced Cardiotoxicity in Rats and Potentiates Its Anticancer Activity on MCF-7 Cells.

Cardiotoxicity is a limiting factor of doxorubicin (DOX)-based anticancer therapy. Due to its beneficial effects, we investigated whether standardized extract of Melissa officinalis (MO) can attenuate doxorubicin-induced cardiotoxicity and can potentiate the efficacy of DOX against human breast cancer cells. MO was administered orally to male albino rats once daily for 10 consecutive days at doses of 250, 500 and 750 mg/kg b.wt. DOX (15 mg/kg b.wt. i.p.) was administered on the 8th day. MO protected against DOX-induced leakage of cardiac enzymes and histopathological changes. MO ameliorated DOX-induced oxidative stress as evidenced by decreasing lipid peroxidation, protein oxidation and total oxidant capacity depletion and by increasing antioxidant capacity. Additionally, MO pretreatment inhibited inflammatory responses to DOX by decreasing the expressions of nuclear factor kappa-B, tumor necrosis factor-alpha and cyclooxygenase-2 and the activity of myeloperoxidase. MO ameliorated DOX-induced apoptotic tissue damage in heart of rats. In vitro study showed that MO augmented the anticancer efficacy of DOX in human breast cancer cells (MCF-7) and potentiated oxidative damage and apoptosis. Thus, combination of DOX and MO may prove future cancer treatment protocols safer and more efficient.

Differential effects of methoxyamine on doxorubicin cytotoxicity and genotoxicity in MDA-MB-231 human breast cancer cells.

Pharmacological inhibition of DNA repair is a promising approach to increase the effectiveness of anticancer drugs. The chemotherapeutic drug doxorubicin (Dox) may act, in part, by causing oxidative DNA damage. The base excision repair (BER) pathway effects the repair of many DNA lesions induced by reactive oxygen species (ROS). Methoxyamine (MX) is an indirect inhibitor of apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional BER protein. We have evaluated the effects of MX on the cytotoxicity and genotoxicity of Dox in MDA-MB-231 metastatic breast cancer cells. MX has little effects on the viability and proliferation of Dox-treated cells. However, as assessed by the cytokinesis-block micronucleus assay (CBMN), MX caused a significant 1.4-fold increase (P<0.05) in the frequency of micronucleated binucleated cells induced by Dox, and also altered the distribution of the numbers of micronuclei. The fluorescence probe dihydroethidium (DHE) indicated little production of ROS by Dox. Overall, our results suggest differential outcomes for the inhibition of APE1 activity in breast cancer cells exposed to Dox, with a sensitizing effect observed for genotoxicity but not for cytotoxicity.

Synthesis and biological activity of 6-selenocaffeine: potential modulator of chemotherapeutic drugs in breast cancer cells.

We report the development of a new microwave-based synthetic methodology mediated by Woollins' reagent that allowed an efficient conversion of caffeine into 6-selenocaffeine. A preliminary evaluation on the modulation of antioxidant activity upon selenation of caffeine, using the DPPH assay, indicated a mild antioxidant activity for 6-selenocaffeine, contrasting with caffeine, that exhibited no antioxidant activity under the same experimental conditions. Interestingly, whereas 6-selenocaffeine has revealed to have a low cytotoxic potential in both MCF10A and MCF-7 breast cells (24 h, up to 100 µM, MTT assay), a differential effect was observed when used in combination with the anticancer agents doxorubicin and oxaliplatin in MCF-7 breast cancer cells. The co-treatment of doxorubicin (1 µM) and 6-selenocaffeine (100 µM) resulted in a slight decrease in cellular viability when compared to doxorubicin (1 µM) alone. Conversely, the seleno-caffeine derivative at the same concentration markedly increased the viability of oxaliplatin (100 µM)-treated cells (p < 0.01). Overall, this work highlights an emerging methodology to synthesize organoselenium compounds and points out the differential roles of 6-selenocaffeine in the modulation of the cytotoxicity of anticancer agents.

Synergistic action of the novel HSP90 inhibitor NVP-AUY922 with histone deacetylase inhibitors, melphalan, or doxorubicin in multiple myeloma.

Heat shock protein 90 (HSP90) is a promising target for tumor therapy. The novel HSP90 inhibitor NVP-AUY922 has preclinical activity in multiple myeloma, however, little is known about effective combination partners to design clinical studies. Multiple myeloma cell lines, OPM-2, RPMI-8226, U-266, LP-1, MM1.S, and primary myeloma cells were exposed to NVP-AUY922 and one of the combination partners histone deacetylase inhibitor NVP-LBH589, suberoylanilide hydroxamic acid (SAHA), melphalan, or doxorubicin, either simultaneously or in sequential patterns. Effects on cell proliferation and apoptosis were determined. Synergistic effects were evaluated using the method of Chou and Talalay. Combined sequential incubation with NVP-AUY922 and SAHA showed that best synergistic effects were achieved with 24 h preincubation with SAHA followed by another 48 h of combination treatment. Combination of NVP-AUY922 with SAHA, NVP-LBH589, melphalan, or doxorubicin resulted in synergistic inhibition of viability, with strong synergy (combination index < 0.3) in the case of melphalan. Importantly, resistance of the RPMI-8226 cell line and relative resistance of some primary myeloma cells against NVP-AUY922 could be overcome by combination treatment. These data show impressive synergistic action of the novel HSP90 inhibitor NVP-AUY922 with melphalan, doxorubicin, NVP-LBH589, and SAHA in multiple myeloma and build the frame work for clinical trials.

Apoptosis-inducing effects of Morinda citrifolia L. and doxorubicin on the Ehrlich ascites tumor in Balb-c mice.

Morinda citrifolia L. (Noni) is a herbal remedy with promising anti-cancer properties. However, its effects on various cancers are to be investigated to make a firm conclusion before implementing it into the clinical practice. Therefore, we investigated the cytotoxic potential of noni on Ehrlich ascites tumor grown in female Balb-c mice and also combined it with a potent anti-cancer agent, doxorubicin. One group received noni only (n = 8), another one doxorubicin (n = 8), and the other one noni + doxorubicin (n = 8) for 14 days after the inoculation of cells. The control group (n = 7) received 0.9% NaCl only. We found that short and long diameters of the tumor tissues were about 40-50% smaller, compared to those in control group. This anti-growth effect resulted from the induction of apoptosis, which was confirmed by the positive results from the Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) analysis and the active caspase-3 cells in tissues. Apoptosis also confirmed by caspase-cleaved cytokeratin 18 elevation in serum of the treated groups. Further, the proliferation was decreased, which was immunohistochemically shown by the PCNA staining. We conclude that noni may be useful in the treatment of breast cancer either on its own or in combination with doxorubicin. Further studies are warranted to assess the dosage and safety of using noni fruit juice in conjuction with anti-cancer drugs against breast cancer.

Suppression of PTEN function increases breast cancer chemotherapeutic drug resistance while conferring sensitivity to mTOR inhibitors.

Ectopic expression of mutant forms of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) lacking lipid (G129E) or lipid and protein (C124S) phosphatase activity decreased sensitivity of MCF-7 breast cancer cells, which have wild-type PTEN, to doxorubicin and increased sensitivity to the mammalian target of rapamycin (mTOR) inhibitor rapamycin. Cells transfected with a mutant PTEN gene lacking both lipid and protein phosphatase activities were more resistant to doxorubicin than cells transfected with the PTEN mutant lacking lipid phosphatase activity indicating that the protein phosphatase activity of PTEN was also important in controlling the sensitivity to doxorubicin, while no difference was observed between the lipid (G129E) and lipid and protein (C124S) phosphatase PTEN mutants in terms of sensitivity to rapamycin. A synergistic inhibitory interaction was observed when doxorubicin was combined with rapamycin in the phosphatase-deficient PTEN-transfected cells. Interference with the lipid phosphatase activity of PTEN was sufficient to activate Akt/mTOR/p70S6K signaling. These studies indicate that disruption of the normal activity of the PTEN phosphatase can have dramatic effects on the therapeutic sensitivity of breast cancer cells. Mutations in the key residues which control PTEN lipid and protein phosphatase may act as dominant-negative mutants to suppress endogenous PTEN and alter the sensitivity of breast cancer patients to chemo- and targeted therapies.

The tumor-growth inhibitory activity of flavanone and 2'-OH flavanone in vitro and in vivo through induction of cell cycle arrest and suppression of cyclins and CDKs.

Natural products, including flavonoids, are suggested to be involved in the protective effects of fruits and vegetables against cancer. However, studies concerning the effect of flavonoids frequently lacked data regarding to flavanones. In this study, we investigated the inhibitory effect of flavanone compounds, including flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone, naringin and naringenin, on cell growth of various cancer cells. We determined that flavanone and 2'-OH flavanone inhibited cell growth of A549, LLC, AGS, SK-Hepl and HA22T cancer cells, while other flavanones showed little or no inhibition. We evaluated growth-inhibitory activity of flavanone and 2'-OH flavanone against highly proliferative human lung cancer cells (A549) via anchorage-independent and -dependent colony formation assay, and further showed that treatment of flavanone resulted in a G1 cell cycle arrest with reduction of cyclin D, E and cyclin-dependent kinase (CDK) 2, while treatment of 2'-OH flavanone led to a G2/M phase accumulation with reduction of cyclin B, D and Cdc2. Moreover, we demonstrated the improvement effect of flavanone and 2'-OH flavanone with anti-cancer drug, doxorubicin, on A549 cells. Finally, flavanone and 2'-OH flavanone were evidenced by its inhibition on the growth of A549 and Lewis lung carcinoma cells in vivo.

Proanthocyanidin from grape seeds enhances doxorubicin-induced antitumor effect and reverses drug resistance in doxorubicin-resistant K562/DOX cells.

With the aim of enhancing the efficacy of chemotherapeutic agents, we investigated the antitumor actions and reversal effect on drug resistance of proanthocyanidin plus doxorubicin. The results showed that proanthocyanidin 12.5-200 mg/L significantly inhibited proliferation of K562, K562/DOX, SPC-A-1, and Lewis cells in vitro in a time- and concentration-dependent manner, as determined by microculture tetrazolium assay. A combination of proanthocyani din 12.5, or 25 mg/L and doxorubicin treatment synergistically inhibited cell proliferation with decreased IC50 values. Proanthocyanidin reverses drug resistance in doxorubicin-resistant K562/DOX cells, and IC50 values were decreased by 9.19 (3.64-23.19), 2.56 (1.48-.44), and 0.94 (0.81-1.09) mg/L, respectively, after 24 h treatment with doxorubicin 0.1-9.0 mg/L alone or in combination with proanthocyanidin 12.5 or 25 mg/L; the proanthocyanidin reversal fold was 3.6 and 9.8, respectively. Under confocal laser scanning microscope, the combination of proanthocyanidin 25 or 50 mg/L with doxorubicin 3 mg/L significantly increased the accumulation of intracellular doxorubicin, Ca2+, and Mg2+, and reduced the pH value and mitochondrial membrane potential in K562/DOX cells as compared with doxorubicin alone (p < 0.01). Additionally, the apoptosis rate was increased by 11.3% +/- 3.3%, 14.2% +/- 5.4%, and 23.8% +/- 2.8%, respectively, for doxorubicin 3 mg/L alone or with proanthocyanidin 12.5 or 25 mg/L, as compared with controls (3.0% +/- 1.4%), as demonstrated by flow cytometry. In vivo experiments demonstrated that i.p. administration of proanthocyanidin 10 mg/kg with doxorubicin 2 mg/kg had an inhibitory effect on the growth of transplantation tumor sarcoma 180 and hepatoma 22 in mice as compared with doxorubicin alone (p < 0.05). These results suggest that proanthocyanidin enhances doxorubicin-induced antitumor effect and reverses drug resistance, and its mechanism is attributed partially to the promotion of doxorubicin-induced apoptosis through an elevation of intracellular doxorubicin, and Ca2+, Mg2+ concentration, and a reduction of pH value and mitochondrial membrane potential.