Dose Reduction Index Research Articles

Carfilzomib in Combination with Bortezomib Enhances Apoptotic Cell Death in B16-F1 Melanoma Cells.

Simple SummaryThe incidence rate of metastatic melanoma has been rapidly increasing worldwide and its 5-year survival rate is very low. Due to partial responses, various side effects, and resistance to any known cancer therapeutics, more potent and safer therapeutics are needed to increase the survival rate of patients with melanoma. Since proteasome inhibitors, such as bortezomib and carfilzomib, have been suggested as treatments for various cancers, we investigated their potential for the treatment of melanoma by studying their molecular mechanisms of action in B16-F1 melanoma cells. In this study, we found that both bortezomib and carfilzomib lead to apoptosis via ER stress as well as ROS accumulation and MMP loss in melanoma cells. Bortezomib and carfilzomib synergistically reduced B16-F1 tumor growth in vitro and in a C57BL/6 xenograft mouse model. Therefore, a combination therapy with carfilzomib and bortezomib at submaximal concentrations may reduce their side effects and be beneficial for melanoma treatment.Proteasome inhibitors, such as bortezomib (BZ) and carfilzomib (CFZ), have been suggested as treatments for various cancers. To utilize BZ and/or CFZ as effective therapeutics for treating melanoma, we studied their molecular mechanisms using B16-F1 melanoma cells. Flow cytometry of Annexin V-fluorescein isothiocyanate-labeled cells indicated apoptosis induction by treatment with BZ and CFZ. Apoptosis was evidenced by the activation of various caspases, including caspase 3, 8, 9, and 12. Treatment with BZ and CFZ induced endoplasmic reticulum (ER) stress, as indicated by an increase in eIF2α phosphorylation and the expression of ER stress-associated proteins, including GRP78, ATF6α, ATF4, XBP1, and CCAAT/enhancer-binding protein homologous protein. The effects of CFZ on ER stress and apoptosis were lower than that of BZ. Nevertheless, CFZ and BZ synergistically induced ER stress and apoptosis in B16-F1 cells. Furthermore, the combinational pharmacological interactions of BZ and CFZ against the growth of B16-F1 melanoma cells were assessed by calculating the combination index and dose-reduction index with the CompuSyn software. We found that the combination of CFZ and BZ at submaximal concentrations could obtain dose reduction by exerting synergistic inhibitory effects on cell growth. Moreover, this drug combination reduced tumor growth in C57BL/6 syngeneic mice. Taken together, these results suggest that CFZ in combination with BZ may be a beneficial and potential strategy for melanoma treatment.

Selective ERBB2 and BCL2 Inhibition Is Synergistic for Mitochondrial-Mediated Apoptosis in MDS and AML Cells.

The ERBB2 proto-oncogene is associated with an aggressive phenotype in breast cancer. Its role in hematologic malignancies is incompletely defined, in part because ERBB2 is not readily detected on the surface of cancer cells. We demonstrate that truncated ERBB2, which lacks the extracellular domain, is overexpressed on primary CD34+ myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cells compared with healthy hematopoietic cells. This overexpression of ERBB2 is associated with aberrant, oncogenic signaling with autophosphorylation of multiple tyrosine sites. Like in breast cancers, ERBB2 can exist as truncated isoforms p95ERBB2 and p110ERBB2 in MDS and AML. Neutralization of ERBB2 signaling with ERBB2 tyrosine kinase inhibitors (i.e., lapatinib, afatinib, and neratinib) increases apoptotic cell death and reduces human engraftment of MDS cells in mice at 21 weeks posttransplantation. Inhibition of ERBB2 modulates the expression of multiple pro- and anti-apoptotic mitochondrial proteins, including B-cell lymphoma 2 (BCL2). Dual blockade with ERBB2 and BCL2 inhibitors triggers additional reductions of BCL2 phosphorylation and myeloid cell leukemia-1 (MCL1) expression compared with single drug treatment. Dual therapy was synergistic at all tested doses, with a dose reduction index of up to 29 for lapatinib + venetoclax compared with venetoclax alone. Notably, these agents operated together and shifted cancer cells to a pro-apoptotic phenotype, resulting in increased mitochondrial cytochrome c release and activated caspase-3-mediated cell death. IMPLICATIONS: These findings warrant study of ERBB2 and BCL2 combination therapy in patients with MDS and AML. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/5/886/F1.large.jpg.

1α,25-Dihydroxyvitamin D3 synergistically enhances anticancer effects of ginsenoside Rh2 in human prostate cancer cells

1α,25-dihydroxyvitamin D3 (1,25(OH)2D3, commonly known as calcitriol), the most active metabolite of vitamin D3, and ginsenoside Rh2 can regulate cellular differentiation and proliferation proteins. The purpose of the present study was to assess the effect of 1,25(OH)2D3 on the anticancer activities of Rh2 in human prostate cancer cells such as androgen-dependent LNCaP and androgen-independent C4-2 in vitro. The effects of treatment with 1,25(OH)2D3 or Rh2, either alone or in combination, on prostate cancer cells were evaluated through tetrazolium-based cell viability assay, BrdU cell proliferation rate estimation assay, and Western blot protein expression analyses of nuclear receptors (androgen receptor and vitamin D receptors) and apoptotic proteins (Bcl-2, Bax, and Caspase 3). The Combination Indices (CI) and Dose Reduction Indices (DRI) of 1,25(OH)2D3 and Rh2 were calculated to determine synergistic anticancer activity using Calcusyn software (Biosoft, Cambridge, UK). The cell viability assay data indicate that Rh2 treatment alone inhibited cell viability in a concentration-dependent manner and the addition of 10 nM 1,25(OH)2D3 to Rh2 significantly enhanced its ability to reduce cell viability up to 80 % in both the cell lines. Similarly, addition of 10 nM 1,25(OH)2D3 to Rh2 significantly lowered its IC50 values for cell proliferation from the range of 32–65 μM to 14-8 μM in LNCaP and C4-2 cells. In addition, protein expression analyses indicated that the combined treatment with Rh2 and 1,25(OH)2D3 led to greater downregulation of androgen receptor expression compared to single agent exposure. Similarly, the presence of 1,25(OH)2D3 synergistically increased the pro-apoptotic actions of Rh2 in both the cell lines. Overall, 1,25(OH)2D3 augments the Rh2-mediated anticancer effects through stimulating apoptosis and reduced cell proliferation which suggests that synergism of this combination may lead to potential lower need of the active vitamin D3 and limited toxicity from it.

An Arthropod Hormone, Ecdysterone, Inhibits the Growth of Breast Cancer Cells via Different Mechanisms.

Ecdysterone (Ecdy) is a hormone found in arthropods, which regulates their development. It is also synthesized by a number of plants to combat insect pests. It provides a number of beneficial pharmacological effects including the anabolic and adaptogenic ones. Ecdysterone is widely marketed as food supplement to enhance the physical performance of athletes. In addition to the estrogen receptor beta (ERbeta)-dependent anabolic effect of Ecdy in muscles, the molecular mechanisms of the plethora of other Ecdy-induced pharmacological effects remain unknown. The aim of this study was to investigate the pharmacological effect of ecdysterone on human breast cancer cell lines of different molecular subtypes. Surprisingly, in contrast to the anabolic effect on muscle tissues, we have revealed a tumor suppressive effect of Ecdy on a panel of breast cancer cell lines studied. Using the SeaHorse-based energy profiling, we have demonstrated that Ecdy dampened glycolysis and respiration, as well as greatly reduced the metabolic potential of triple negative breast cancer cell lines. Furthermore, we have revealed that Ecdy strongly induced autophagy. As part of the combined treatment, based on the Combination Index (CI) and Dose Reduction Index (DRI), Ecdy synergized with doxorubicin to induce cell death in several breast cancer cell lines. In contrast, Ecdy had only minor effect on non-transformed human fibroblasts. Collectively, our results indicate that ecdysterone can be considered as a new potential adjuvant for genotoxic therapy in treatment of breast cancer patients.

Cannabinoid Combination Induces Cytoplasmic Vacuolation in MCF-7 Breast Cancer Cells.

This study evaluated the synergistic anti-cancer potential of cannabinoid combinations across the MDA-MB-231 and MCF-7 human breast cancer cell lines. Cannabinoids were combined and their synergistic interactions were evaluated using median effect analysis. The most promising cannabinoid combination (C6) consisted of tetrahydrocannabinol, cannabigerol (CBG), cannabinol (CBN), and cannabidiol (CBD), and displayed favorable dose reduction indices and limited cytotoxicity against the non-cancerous breast cell line, MCF-10A. C6 exerted its effects in the MCF-7 cell line by inducing cell cycle arrest in the G2 phase, followed by the induction of apoptosis. Morphological observations indicated the induction of cytoplasmic vacuolation, with further investigation suggesting that the vacuole membrane was derived from the endoplasmic reticulum. In addition, lipid accumulation, increased lysosome size, and significant increases in the endoplasmic reticulum chaperone protein glucose-regulated protein 78 (GRP78) expression were also observed. The selectivity and ability of cannabinoids to halt cancer cell proliferation via pathways resembling apoptosis, autophagy, and paraptosis shows promise for cannabinoid use in standardized breast cancer treatment.

Curcumin and wikstroflavone B, a new biflavonoid isolated from Wikstroemia indica, synergistically suppress the proliferation and metastasis of nasopharyngeal carcinoma cells via blocking FAK/STAT3 signaling pathway

BackgroundCurcumin (CUR) is a natural diarylheptanoid with marked anti-tumor activities. Recent investigations demonstrate that CUR combines with some other phytochemicals exerts advantages over its single application manifested as lower toxicity, higher efficacy or more significant reversal of multidrug resistance. PurposeThis study aimed to elucidate a new biflavonoid (wikstroflavone B, WFB) isolated from Wikstroemia indica and to assess the synergistic inhibition of combined CUR and WFB (CUR/WFB) on human nasopharyngeal carcinoma (NPC) cell lines proliferation and metastasis. MethodsWFB was obtained through sequential chromatographic methods including silica gel, Sephadex LH-20 and preparative HPLC. Its structure was determined by HRESIMS, 1D and 2D NMR spectroscopic analysis. The absolute configuration of WFB was assigned through comparison of experimental and calculated optical rotation (OR) values. Changes in cellular viability, migration and invasion were assessed by MTT, colony formation, wound healing and Transwell assays. The nature of synergistic interaction of CUR/WFB was determined through the combination index (CI) method under the median-effect analysis. Expression levels of indicated mRNAs and proteins were measured by qRT-PCR and Western blotting assays, respectively. ResultsWFB was isolated and structural elucidated. Compared with CUR or WFB used alone, CUR/WFB treatment inhibited more effectively on the cell viability, colony formation, cell migration and invasion. Both CI and dose reduction index (DRI) values indicated the significant synergistic effects existed between CUR and WFB. Besides, CUR/WFB showed the marked modulation on the genes involved in cell proliferation (survivin, cyclin D1, p53 and p21) and metastasis (MMP-2, MMP-9 and FAK). CUR/WFB treatment was also found to restrain the phosphorylation of FAK and STAT3 proteins. When pretreatment with a FAK inhibitor, the cell viability and metastasis were significantly attenuated. ConclusionThe results indicate that WFB can synergistically increase the inhibitory effects of CUR on NPC cells proliferation and metastasis, and these findings may afford a rational approach for developing the antitumor medications.

Investigation of synergistic potential of green tea polyphenols and orlistat combinations using pancreatic lipase assay-based synergy directed fractionation strategy

The present study investigated the synergistic potential of methanol extract/fractions of Camellia sinensis (L.) Kuntze (CSM) leaves and orlistat on inhibition of Pancreatic Lipase (PL). Since PL is responsible for 50–70% of dietary fats digestion, its inhibition is considered as a promising target for the management of obesity. A synergy guided fractionation strategy has been incorporated with the help of combination index (CI) and isobologram methods. CSM exhibited a potential synergistic activity in combination with orlistat (CI value: 0.66). Amongst the subfractions of CSM, polyphenol-rich fraction exhibited a greater synergistic potential (CI-0.35) than remaining fractions. HPLC analysis identified epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) as major phytochemicals in polyphenol-rich fraction and the most active subfractions (F6 and F7). Two drug and three-drug combinations of these phytochemicals were investigated by partnering with orlistat. The three-drug combination (orlistat-EGCG-ECG) exhibited a greater synergistic potential (CI value-0.17) than the two-drug combinations of EGCG-orlistat and ECG-orlistat (CI values of 0.40 & 0.71, respectively). The resultant orlistat-EGCG-ECG combination resulted in a 12.11-fold dose reduction index for orlistat. Thus, synergistic drug combinations of polyphenol-rich fraction along with orlistat offers the prospect to enhance the orlistat anti-obesity efficacy in a decreased dose level, that will help to reduce its associated toxicities while maintaining its efficacy.

Abstract 1907: Src-Met signaling confers apoptosis resistance to EGFR and PI3K co-targeting independently of AKT and ERK pathway activation in head and neck cancer

Abstract Purpose: Molecularly targeted agents will play a major role in the next generation of personalized cancer therapies. However, intrinsic and acquired resistance to targeted agents poses challenges to the success of such treatments. The purpose of the current study was to investigate the intrinsic resistance of head and neck cancer (HNC) cell lines to apoptosis induced by the combination of EGFR inhibitor erlotinib and PI3K inhibitor BKM120. Methods: In a panel of 10 malignant and 1 premalignant HNC cell lines, we evaluated cell growth inhibition (by SRB assay), IC50, combination index, dose reduction index (by CalcuSyn Software), and apoptosis (by Annexin-V staining). Protein expression levels were measured by Western blotting. Small molecule inhibitors and siRNA-mediated knockdown strategies were used to inactivate and shut down the expression of the relevant proteins, respectively. Results: We have reported previously that single targeting of EGFR with erlotinib or PI3K with BKM120 (pan-PI3K inhibitor) suppressed cellular growth without inducing significant apoptosis. Co-targeting EGFR and PI3K demonstrated in vitro synergy in all except the JHU022 cell line and more effectively inhibited HNC xenograft growth in vivo. The combination of erlotinib and BKM120 induced variable apoptosis: some cell lines were very sensitive (MDA686TU, MDA686LN, 93-VU-147T), some moderately (Fadu, SqCCy1, MDA1483, UPCI:SCC090) and others were resistant (UD-SCC2, MSK-LEUK1, JHU022) to apoptosis induction. We found that the resistant cell line JHU022 expressed very high levels of p-Met (Y1234/1235). Inhibition of Met with small molecule inhibitor crizotinib or siRNA rendered this cell line highly sensitive to apoptosis induced by the combination of erlotinib and BKM120. Although inhibition of hepatocyte growth factor (HGF), the only known ligand for Met, with soluble neutralization antibody had no effect on Met phosphorylation, inhibition of Src family kinases (SFK) with dasatinib abolished Met phosphorylation at Y1234/1235. Dasatinib also made this cell line sensitive to apoptosis. Ablation of c-Src with siRNA had similar effects on p-Met (Y1234/1235) and apoptosis. Interestingly, inhibition of Met had no effects on AKT and ERK phosphorylation, two critical downstream pathways of receptor tyrosine kinases including Met. Conclusions: Our results strongly suggest that co-targeting of EGFR and PI3K has synergistic activity, is effective in vivo, and induces apoptosis of some HNC cell lines. Some resistant cell lines express a high level of p-Met as a downstream target of SFK, which confers resistance of these cells to EGFR and PI3K co-targeting. Finally, Met confers apoptosis resistance independently of AKT and ERK pathway activation. Citation Format: Abu S. Anisuzzaman, Abedul Haque, Zhuo G. Chen, Dong M. Shin, A.R.M. Ruhul Amin. Src-Met signaling confers apoptosis resistance to EGFR and PI3K co-targeting independently of AKT and ERK pathway activation in head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1907.

Abstract 4119: Necroptosis inducing thienopyridine analogs overcomes chemoresistance in TNBC

Abstract Triple-negative breast cancer (TNBC), defined by the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2), is the most aggressive and lethal form of breast cancer. The treatment of chemo-resistant TNBC tumors is particularly challenging due to the overexpression of efflux transporters, increased DNA repair, and the occurrence of genetic mutations that decrease the likelihood of apoptosis. Lack of clear targets and development of drug resistance allows TNBC to advance to metastatic stage thus resulting in poor prognosis and survival outcome. Recently, we discovered thieno-pyrimidin-hydrazinyl (TPH) class of small molecules that causes necroptotic (i.e. programmed necrosis) cell death in TNBC cells. We found that the lead compound TPH104 has an IC50 value of 160-400 nm and has ~400-fold selectivity towards different TNBC cell lines compared to normal breast cells. TPH104 selectively induced receptor interacting protein kinase 1 (RIPK1) - mediated necroptosis, while promoting autophagy thus resulting in significant inhibition of TNBC cell proliferation. We characterized eighteen novel TPH analogs for their cytotoxic potential and structure activity relation (SAR) and learned that the subtle changes in TPH pharmacophore could enhance or decrease efficacy against TNBC cells. We observed that 2-OH group is required to exert cytotoxic potency. Two new hit analogs of TPH104, TPH104c and TPH104m were found to activate necroptotic cell death markers (RIP, MLKL) with IC50 values similar to TPH104 in TNBC cells. Both analogs decreased the TNBC cell proliferation, and rate of colony formation compared to controls. Mechanistically, similar to TPH104, these analogs did not produce significant apoptosis induction i.e. no loss of mitochondrial membrane potential and arrested the TNBC cells in S-phase of cell cycle. Interestingly, unlike TPH104, TPH104c and TPH104m activate executioner caspases 3 and 7 only at extremely high concentration. TPH104 and its analogs reversed resistance mediated by ABCB1 and ABCG2 transporters through collateral sensitivity. TPH analogs in combination with doxorubicin and paclitaxel synergistically inhibited TNBC and TNBC/resistant (R) cells and reduced the dose-reduction index. Further understanding SAR of TPH analogs and the biology of non-apoptotic, necroptosis cell death by this new class of small molecules will help define a multimodal, non-apoptotic approach to overcome chemoresistance in TNBC patients. This will lead to the development of more selective and efficacious compounds that kills TNBC cells through unique cell death mechanism. Citation Format: Saloni Malla, Diwakar Bastihalli Tukaramrao, Divya L. Dayanidhi, Pia Vogel, Shikha Kumari, Amit K. Tiwari. Necroptosis inducing thienopyridine analogs overcomes chemoresistance in TNBC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4119.

Abstract A23: Verteporfin as a new treatment paradigm for platinum-resistant ovarian cancer cells

Abstract Ovarian cancer is the most lethal of the gynecologic malignancies and has poor survival rates due to a combination of diagnosis at advanced stages and disease recurrence due to platinum chemotherapy resistance. It is of utmost importance that novel chemotherapeutic agents are identified to increase or restore chemosensitization to platinum-based chemotherapy. Verteporfin (VP, VisudyneTM) is a benzoporphyrin derivative used for treating adult macular degeneration. Verteporfin has recently been identified as an inhibitor of YAP/TEAD interaction. Yes-associated protein (YAP), the main downstream target of the HIPPO pathway, plays an important role in cell proliferation and apoptosis. We investigated the efficacy of VP alone or in combination with cisplatin (CDDP) or carboplatin (CP) or paclitaxel (Taxol) on two serous ovarian cancer cell lines (OV90, COV504). The effect of VP, CDDP, CP or Taxol was studied either alone or in combination after 72 hours of treatment. Cell viability-based cytotoxicity measurements indicate dose-dependent increase in cytotoxicity for all the drugs. IC50 values of the drugs were calculated based on dose-effect curves using Compusyn software following the Chou-Talalay method. For OV90 cells, the calculated IC50 values were 29.33μM for VP, 2.17μM for cisplatin, 96.21μM for carboplatin, and 89.05μM for paclitaxel. For COV504 cells, the calculated IC50 values were 8.37μM for VP, 1.79μM for cisplatin, 117.23μM for carboplatin, and 7.16μM for paclitaxel. Based on the IC50 values of single agents, we performed cell proliferation assay using VP and CDDP/CP/Taxol in combination. We observed synergistic effects of VP with CDDP/CP/Taxol based on combination index plots. Based on the dose-reduction index (DRI) values of the drugs in combination treatments, we performed cytotoxicity and invasion assays. The combination treatments of these drugs at DRI values induced cytotoxicity and inhibited invasion of cancer cells. To check the chemosensitization of VP in cancer cells, we used cisplatin-resistant ovarian cancer cells, A2780Cis. Similar to ovarian cancer cells, VP effectively inhibited proliferation of A2780Cis cells. Based on these results, we hypothesize that VP is inducing chemosensitivity in platinum resistant ovarian cancer cells and increasing the efficacy of either platin drugs or Taxol. We suggest that our preclinical data in combination with previously available human safety data support the study of VP in combination with platinum in a clinical phase 2 trial of serous ovarian cancer patients. Note: This abstract was not presented at the conference. Citation Format: Radhika P. Gogoi, Venkata Ramesh Dasari. Verteporfin as a new treatment paradigm for platinum-resistant ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A23.

Synergic Effect of Curcuma longa L. Extract with Antimicrobials against Cuban Helicobacter pylori Isolates

AimsHelicobacter pylori is a bacterium that colonizes the gastric mucosa and is the main cause of gastritis as well as ulcer and gastric cancer. Due to the clinical significance and international increase in H. pylori multidrug resistance, it is necessary to search for new strategies improving eradication rates. Natural compounds have been demonstrated to have antimicrobial effect and the ability to restore the efficacy of conventional drugs. The objective of this work was to evaluate the antimicrobial effect of the hydroalcoholic extract of Curcuma longa L. (Cu) against H. pylori isolates.Materials & MethodsThe minimum bactericidal concentration of the extract was determined by means of the MTT assay; also, the combination and dose reduction indices for levofloxacin (LVX), metronidazole (MET), and rifampicin (RIF) antimicrobial agents were determined by checkerboard format. Interaction analysis was performed using the CompuSyn program.FindingsAbout 90% of H. pylori isolates studied (9/10) were sensitive to the hydroalcoholic extract. Synergism was observed in more than 50% of Cu-LVX, Cu-MET, and Cu-RIF combinations. Additionally, for different concentrations of the extract, reduction rates in antimicrobial agents were determined to be between 0.5 and 360 times.ConclusionsThe hydroalcoholic extract of turmeric showed a good potential to be used as an antimicrobial agent in the treatment of H. pylori infection, either alone or in combination with antibiotics used, suggesting the renewal of the effectiveness of conventional antimicrobials in reducing the phenomenon of antimicrobial resistance.

The use of eugenol in combination with cefotaxime and ciprofloxacin to combat ESBL-producing quinolone-resistant pathogenic Enterobacteriaceae.

Emergence of extended-spectrum beta-lactamase (ESBL) producing with quinolone-resistant (QR) pathogenic Enterobacteriaceae augmented the need to establish therapeutic options against them. Present study aimed towards determination of synergistic combination of eugenol (EG) with cefotaxime (CTX) and ciprofloxacin (CIP) to combat against this resistance and potentiation of antibacterial drugs by EG against these bacteria. Synergistic interaction between EG and CTX/CIP (FICI: 0·08-0·5) were observed among ESBL-QR bacteria using checkerboard assay. Approximately, 2- to 1024-fold minimum inhibitory concentration value reduction and 17- to 165030-fold dose reduction index strongly suggested synergistic interaction between EG and antibiotics. Cell viability assay showed reduction in log10 CFU per ml from 16·6 to 3·1 at synergistic concentration. Scanning electron microscopy further proved disruptive effect of EG on cell architecture. Eugenol and/or its combination also altered genes' expressions that imparted antibiotic resistance by ~1·6 to ~1226 folds. Reduced doses of antibiotics, bacterial morphological alterations, efflux pump down regulation, porin over expression and beta-lactamase gene inhibition of ESBL-QR bacteria by EG alone or in combination with CTX/CIP might have reversed antibiotic resistance profile of ESBL-QR bacteria. This study provided a molecular insight into action of EG and/with CTX and CIP, which might have potentiated antibiotic's activity against ESBL-QR bacteria.

Synergistic combinations of paclitaxel and withaferin A against human non-small cell lung cancer cells.

Platinum-taxane combination chemotherapy still represents the standard of care for advanced non-small cell lung cancer (NSCLC) with no targetable driver mutations. However, the efficacy of these drugs has plateaued at 10–14 months primarily due to dose-limiting toxicity, chemoresistance, and metastasis. Here, we explored the effects of withaferin A (WFA) alone and in combination with paclitaxel (PAC) on the growth, proliferation, migration, and invasion of human NSCLC cells. We show that the sensitivity of H1299 and A549 cells to concomitant treatment with PAC and WFA was greater than that of either PAC or WFA alone. Using the combination index and dose-reduction index, we demonstrated that various combinations (1:40, 1:20, 1:10) of PAC to WFA, respectively, were highly synergistic. In addition, PAC+WFA co-treatment synergistically inhibited colony formation, migration, invasion and increased the induction of apoptosis in H1299 and A549 cells. Interestingly, the synergism of PAC and WFA was not schedule-dependent but was enhanced when cells were pretreated with WFA indicating a chemo-sensitizing effect. Importantly, WFA was active against both PAC-sensitive (TS-A549) and PAC-resistant (TR-A549) cells both in vitro and in vivo. Mechanistically, WFA inhibits the proliferation of NSCLC cells via thiol oxidation. The effects of WFA were inhibited in the presence of N-acetyl cysteine and other thiol donors. Taken together, our results demonstrate the efficacy of WFA alone or alongside PAC on NSCLC cells and provide a strong rationale for further detailed testing in clinically relevant models for the development of PAC+WFA combination as an alternative therapeutic strategy for advanced NSCLC.

Efficacy and cytotoxic potential of deltamethrin, essential oils of Cymbopogon citratus and Cinnamonum camphora and their synergistic combinations against stored product pest, Trogoderma granarium (Everts)

Residual film method was followed to analyze the toxicity of deltamethrin, essential oils (EOs) of Cymbopogon citratus and Cinnamonum camphora in separate and form of various combinations against LHR1 and LHR2 populations of fifth instar larvae of Trogoderma granarium. The cytotoxic potential of LC50s of both EOs, deltamethrin and their various combinations was as well investigated. The LHR2 population showed 23.73 fold deltamethrin resistance with respect to LHR1 population. Both EOs had high insecticidal activity than deltamethrin but C. citratus EO was less toxic than C. camphora EO against both populations. Based on strength of synergism and dose reduction index, the order of insecticidal activity of combinations in LHR1 population was: C. citratus EO: deltamethrin < C. camphora EO: deltamethrin < C. citratus EO: C. camphora EO < C. citratus EO: C. camphora EO: deltamethrin. In LHR2 population the order was: C. citratus EO: C. camphora EO < C. citratus EO: deltamethrin < C. camphora EO: deltamethrin < C. citratus EO: C. camphora EO: deltamethrin. Following to exposure of LC50 of each treatment various markers of cytotoxicity viz., haemocyte viability, cell proliferation rate and mitochondrial dehydrogenase activity were decreased significantly with respect to control in both populations. Except for deltamethrin, each treatment significantly increased the lactate dehydrogenase activity. Phenoloxidase activity was reduced significantly in LHR1 population following to exposure of each treatment. Likewise, LHR2 population also showed significantly reduced activity of phenoloxidase following to exposure of each treatment except deltamethrin as it increased the activity. Furthermore, each marker of cytotoxicity was fluctuated in relation to the toxicity of LC50 of treatments and combination contained both EOs and deltamethrin produced highest cytotoxicity. Findings of this study should be used in granaries for the effective control of insecticide resistance in T. granarium1.

Antimicrobial and Antioxidant Properties of Four Lycopus Taxa and an Interaction Study of Their Major Compounds.

The compositions of leaf infusions of three genotypes of Lycopus europaeus L. with origins in central Europe, namely L. europaeus A (LeuA), L. europaeus B (LeuB), and L. europaeus C (LeuC), and one genotype of L. exaltatus (Lex), were examined by LC-MS-DAD (Liquid Chromatography Mass Spectrometry and Diode Array Detection) analysis. This revealed the presence of thirteen compounds belonging to the groups of phenolic acids and flavonoids, with a predominance of rosmarinic acid (RA) and luteolin-7-O-glucuronide (LGlr). The antimicrobial activity of leaf infusions was tested on the collection strains of Gram-positive and Gram-negative bacteria, and on the clinical Staphylococcus aureus strains. We detected higher activity against Gram-positive bacteria, of which the most susceptible strains were those of Staphylococcus aureus, including methicillin-resistant and poly-resistant strains. Furthermore, we examined the antioxidant activity of leaf infusions using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) methods, and on NIH/3T3 cell lines using dichlorodihydrofluorescein diacetate (DCFH-DA). We also studied the mutual interactions between selected infusions, namely RA and/or LGlr. In the mixtures of leaf infusion and RA or LGlr, we observed slight synergism and a high dose reduction index in most cases. This leads to the beneficial dose reduction at a given antioxidant effect level in mixtures compared to the doses of the parts used alone. Therefore, our study draws attention to further applications of the Lycopus leaves as a valuable alternative source of natural antioxidants and as a promising topical antibacterial agent for medicinal use.

The synergistic anti-proliferative effect of the combination of diosmin and BEZ-235 (dactolisib) on the HCT-116 colorectal cancer cell line occurs through inhibition of the PI3K/Akt/mTOR/NF-κB axis.

One of the most lethal malignancies worldwide is colorectal cancer (CRC). Alterations in various signalling pathways, including PI3K-mTOR and NF-κB, have been reported in CRC with subsequent dysregulation of proliferation, apoptosis, angiogenesis and, questionably, autophagy processes. BEZ-235 (dactolisib) is a dual PI3K-mTOR inhibitor with potent anti-tumour activity. However, the observed toxicity of BEZ-235 necessitated the termination of its clinical trials. Hence, we aimed to evaluate the potential long-lasting anti-carcinogenic effects of adding diosmin (DIO, a natural NF-κB inhibitor) to BEZ-235 inHCT-116 CRC cells. The median inhibitory concentrations (IC50s) of BEZ-235 and/or DIO were evaluated in the HCT-116 CRC cell line. Caspase-3 activity was assessed colorimetrically, and p-Akt, NF-κB, CD1, VEGF and LC3B levels were assessed by ELISA. Additionally, LC3-II and P62 gene expression were assessed using qRT-PCR. The observed CIs (combination indices) and DRIs (dose reduction indices) confirmed the synergistic effect of DIO and BEZ-235. Co-administration of both drugs either in combination-1 (1μM for BEZ-235, 250μM for DIO) or in combination-2 (0.51μM for BEZ-235 + 101.99μM for DIO) inhibited the PI3K/Akt/mTOR/NF-κB axis, leading to the induction of apoptosis (via active caspase-3), and the inhibition of proliferation marker (CD1), angiogenesis marker (VEGF), autophagy protein (LC3B) and altered effects on LC3-IIandP62 gene expression. Our results reveal the synergistic chemotherapeutic effects of DIO combined with BEZ-235 in the HCT-116 CRC cell line and encourage future preclinical and clinical studies of this combination with reduced BEZ-235 concentrations to avoid its reported toxicity.

Crystal violet structural analogues identified by in silico drug repositioning present anti-Trypanosoma cruzi activity through inhibition of proline transporter TcAAAP069.

BackgroundCrystal violet (CV) was used for several years in blood banks to eliminate the parasite Trypanosoma cruzi in endemic areas in order to prevent transfusion-transmitted Chagas disease. One mechanism of action described for CV involves inhibition of proline uptake. In T. cruzi, proline is essential for host cell infection and intracellular differentiation among other processes, and can be obtained through the proline permease TcAAAP069.Methodology/Principal findingsCV inhibited proline transporter TcAAAP069 and parasites overexpressing this permease were 47-fold more sensitive to this compound than control parasites. Using CV as reference molecule, loratadine, cyproheptadine, olanzapine and clofazimine were identified as structurally related compounds to CV (structural analogues) by in silico drug repurposing through a similarity-based virtual screening protocol. All these already-approved drugs for clinical use inhibited TcAAAP069 activity with different efficacies and also presented trypanocidal action in epimastigotes, trypomastigotes and amastigotes of the Y, CL Brener and Dm28c T. cruzi strains. Finally, a synergistic effect between benznidazole and the CV chemical analogues was evidenced by combination and dose-reduction indexes values in epimastigotes and trypomastigotes of the Y strain.Conclusions/SignificanceLoratadine, cyproheptadine and clofazimine inhibit TcAAAP069 proline transporter and also present trypanocidal effect against all T. cruzi life stages in strains from three different DTUs. These CV structural analogues could be a starting point to design therapeutic alternatives to treat Chagas disease by finding new indications for old drugs. This approach, called drug repurposing is a recommended strategy by the World Health Organization to treat neglected diseases, like Chagas disease, and combination therapy may improve the possibility of success of repositioned drugs.

Antileishmanial Activity and Synergistic Effects of Amphotericin B Deoxycholate with Allicin and Andrographolide against Leishmania martiniquensis In Vitro.

Leishmania (Mundinia) martiniquensis is a causative agent of visceral leishmaniasis, but in HIV-infected patients both visceral and disseminated cutaneous leishmaniasis are presented. Recurrence of the disease after treatment has been reported in some cases indicating that improved chemotherapy is required. In this study, the susceptibility of L. martiniquensis to Amphotericin B deoxycholate (AmB), allicin, and andrographolide was evaluated and the synergistic effects of allicin or andrographolide combined with AmB against L. martiniquensis intracellular amastigotes in mouse peritoneal exudate macrophages (PEMs) were investigated in vitro for the first time. The results showed that L. martiniquensis was highly susceptible to AmB as expected, but allicin and andrographolide had selectivity index (SI) values greater than 10, indicating promise in both compounds for treatment of host cells infected with L. martiniquensis. Four AmB/allicin combinations presented combination index (CI) values less than 1 (0.58–0.68) for intracellular amastigotes indicating synergistic effects. The combination with the highest dose reduction index (DRI) allowed an approximately four-fold reduction of AmB use in that combination. No synergistic effects were observed in AmB/andrographolide combinations. The data provided in this study leads for further study to develop novel therapeutic agents and improve the treatment outcome for leishmaniasis caused by this Leishmania species.

Mentha Rhizomes as an Alternative Source of Natural Antioxidants.

Unlike its aerial parts, the underground parts of Mentha have so far been studied only marginally. By examining the polyphenolic fingerprint, the antioxidant efficacy and the mutual antioxidant behaviour of mixtures of mint rhizomes, our study presents a modest contribution to addressing this gap. Firstly, we examined the composition of the mint rhizomes: Mentha × piperita cv. ‘Perpeta’ (MPP), M. longifolia (ML), and M. × villosa cv. ‘Snežna’ (MVS). Our LC-MS-DAD analysis revealed the presence of ten compounds belonging to groups of phenolic acids and flavonoids, of which the rosmarinic acid (RA) and lithospermic were most strongly represented. Secondly, we evaluated the antioxidant activity of rhizome infusions by DPPH and ABTS and on NIH/3T3 cell lines by DCFH-DA. Thirdly, we determined, examined, and explained the mutual interactions of rhizome infusions mixtures. While most of the combinations acted additive, synergy was observed in ternary infusion mixtures. The synergic action was also detected in the combination of MPP rhizome infusion and RA in the DCFH-DA test. The combinations of mint rhizomes and rosmarinic acid displayed a high dose-reduction index. This leads to beneficial dose reduction at a given antioxidant effect level in mixtures, compared to the dose of the parts used alone. So far, the pharmaceutical and food industry has not used mint rhizomes in commercial products. Hence, our study draws attention to further applications of the Mentha rhizomes as a valuable alternative source of natural antioxidants.

Synergistic and enhanced anticancer effect of a facile surface modified non-cytotoxic silver nanoparticle conjugated with gemcitabine in metastatic breast cancer cells

The safety and efficacy of metallic nanoparticles was one of the major challenges that limit their use in the treatment of cancer. Nanotechnology is applied in the field of pharmaceutical sciences with focus on improving the therapeutic outcome in various diseases. Recently, many novel nano-formulations comprising two or more drugs were studied to improve their efficacy with better safety profile. In this study, we investigated the synergistic cytotoxic effect of gemcitabine (GEM) conjugated non-cytotoxic dose of silver nanoparticles (AgNP) in MDA-MB-453 human triple-negative metastatic breast cancer cells. Synthesized AgNP and electrostatic conjugates were characterized by UV-spectroscopy, dynamic light scattering (DLS), Transmission electron microscopy (TEM), Scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDAX). GEM-(non-cytotoxic AgNP) conjugated system (IC50 = 37.64 μM) showed better cytotoxic activity in MDA-MB-453 cells when compared to individual treatments of GEM (IC50 = 56.54 μM) or AgNP (IC50 = 71.45 μg/ml). The synergism between the GEM-(non-cytotoxic AgNP) for all the tested doses were evaluated using CompuSyn software. The combination index (CI) of ED50, ED75 and ED90 showed synergism for GEM-(non-cytotoxic AgNP) conjugation. According to the calculated dose reduction index (DRI), it requires 1.70-fold less GEM plus 42.55-fold less AgNP to achieve the same 50 % inhibition at 18.38 (GEM): 1 (AgNP) ratio.