Synergistic Cytotoxicity of Renieramycin M and Doxorubicin in MCF-7 Breast Cancer Cells.
ABSTRACT: Renieramycin M (RM) is a KCN-stabilized tetrahydroisoquinoline purified from the blue sponge Xestospongia sp., with nanomolar IC50s against several cancer cell lines. Our goal is to evaluate its combination effects with doxorubicin (DOX) in estrogen receptor positive MCF-7 breast cancer cells. MCF-7 cells were treated simultaneously or sequentially with various combination ratios of RM and DOX for 72 h. Cell viability was determined using the MTT assay. Synergism or antagonism was determined using curve-shift analysis, combination index method and isobologram analysis. Synergism was observed with pharmacologically achievable concentrations of DOX when administered simultaneously, but not sequentially. The IC95 values of RM and DOX after combination were reduced by up to four-fold and eight-fold, respectively. To gain insights on the mechanism of synergy, real-time profiling, cell cycle analysis, apoptosis assays, and transcriptome analysis were conducted. The combination treatment displayed a similar profile with DNA-damaging agents and induced a greater and faster cell killing. The combination treatment also showed an increase in apoptosis. DOX induced S and G2/M arrest while RM did not induce significant changes in the cell cycle. DNA replication and repair genes were downregulated commonly by RM and DOX. p53 signaling and cell cycle checkpoints were regulated by DOX while ErbB/PI3K-Akt, integrin and focal adhesion signaling were regulated by RM upon combination. Genes involved in cytochrome C release and interferon gamma signaling were regulated specifically in the combination treatment. This study serves as a basis for in vivo studies and provides a rationale for using RM in combination with other anticancer drugs.
Project description:Renieramycin M (RM) is a KCN-stabilized tetrahydroisoquinoline purified from the blue sponge Xestospongia sp., with nanomolar IC50s against several cancer cell lines. Our goal is to evaluate its combination effects with doxorubicin (DOX) in estrogen receptor positive MCF-7 breast cancer cells. MCF-7 cells were treated simultaneously or sequentially with various combination ratios of RM and DOX for 72 h. Cell viability was determined using the MTT assay. Synergism or antagonism was determined using curve-shift analysis, Combination Index method and isobologram analysis. Synergism was observed with pharmacologically achievable concentrations of DOX when administered simultaneously, but not sequentially. The IC95 of RM and DOX after combination were reduced by up to 4-fold and 8-fold, respectively. To gain insights on the mechanism of synergy, real-time profiling, cell cycle analysis, apoptosis assays, and transcriptome analysis were conducted. The combination treatment displayed similar profile with DNA-damaging agents and induced a greater and faster cell killing. The combination treatment also showed an increase in apoptosis. DOX induced S and G2/M arrest while RM did not induce significant changes in the cell cycle. DNA replication and repair genes were commonly downregulated by RM and DOX. p53 signaling and cell cycle checkpoints were regulated by DOX while ErbB/PI3K-Akt, integrin and focal adhesion signaling were regulated by RM upon combination. Genes involved in cytochrome C release and interferon gamma signaling were regulated specifically in the combination treatment. This study serves as basis for in vivo studies and provides a rationale for using RM in combination with other anticancer drugs. We used microarray-based transcriptome profiling to gain insights to the mechanism of action of the individual drugs and the synergistic drug combination Overall design: MCF-7 cells were treated with near IC50 concentrations of RM (6.25 nM), DOX (313 nM) and its combination (RM+DOX) for 60 h. Individual drug treatments and vehicle control were prepared in duplicates, while the combination treatment was prepared in triplicates.
Project description:The first total synthesis of (±)-renieramycin I, which was isolated from the Indian bright blue sponge Haliclona cribricutis, is described. The key step is the selenium oxide oxidation of pentacyclic bis-p-quinone derivative (3) stereo- and regioselectively. We also report a large-scale synthesis of cribrostatin 4 (renieramycin H) via the C3-C4 double bond formation in an early stage based on the Avendaño's protocol, from readily available 1-acetyl-3-(3-methyl-2,4,5-trimethylphenyl)methyl-piperazine-2,5-dione (8) in 18 steps (8.3% overall yield). The synthesis provides unambiguous evidence supporting the original structure of renieramycin I.
Project description:Cancer stem cells (CSCs) are distinct cancer populations with tumorigenic and self-renewal abilities. CSCs are drivers of cancer initiation, progression, therapeutic failure, and disease recurrence. Thereby, novel compounds targeting CSCs offer a promising way to control cancer. In this study, the hydroquinone 5-<i>O</i>-cinnamoyl ester of renieramycin M (CIN-RM) was demonstrated to suppress lung cancer CSCs. CIN-RM was toxic to lung cancer cells with a half-maximal inhibitory concentration of around 15 µM. CIN-RM suppressed CSCs by inhibiting colony and tumor spheroid formation. In addition, the CSC population was isolated and treated and the CSCs were dispatched in response to CIN-RM within 24 h. CIN-RM was shown to abolish cellular c-Myc, a central survival and stem cell regulatory protein, with the depletion of CSC markers and stem cell transcription factors ALDH1A1, Oct4, Nanog, and Sox2. For up-stream regulation, we found that CIN-RM significantly inhibited Akt and consequently decreased the pluripotent transcription factors. CIN-RM also inhibited mTOR, while slightly decreasing p-GSK3β (Ser9) but rarely affected the protein kinase C (PKC) signal. Inhibiting Akt/mTOR induced ubiquitination of c-Myc and promoted degradation. The mechanism of how Akt regulates the stability of c-Myc was validated with the Akt inhibitor wortmannin. The computational analysis further confirmed the strong interaction between CIN-RM and the Akt protein with a binding affinity of -10.9 kcal/mol at its critical active site. Taken together, we utilized molecular experiments, the CSC phenotype, and molecular docking methods to reveal the novel suppressing the activity of this compound on CSCs to benefit CSC-targeted therapy for lung cancer treatment.
Project description:A formal total synthesis of the antitumor marine natural product (rac)-renieramycin?T, which possesses a characteristic ecteinascidin-type A?ring in the renieramycin-saframycin core skeleton, was elaborated. The key steps in the synthesis of (rac)-renieramycin?T are a modified Pictet-Spengler cyclization of dialkylated oxomalonate derivatives and decarboxylation via a monocarboxylic acid derivative followed by stereocontrolled protonation of the enol intermediate. A key intermediate in our previous synthesis of renieramycin?T was used, and the formal synthesis was accomplished in 21?steps from a known piperazine-2,5-dione derivative.
Project description:Chemotherapy plays a key role in breast cancer therapy, but drug resistance and unwanted side effects make the treatment less effective. We propose a new combination model that combines antineoplastic drugs and antimalarials for breast cancer therapy. Cytotoxic effects of two antineoplastic agents alone and in combination with several antimalarials on MCF-7 tumor cell line was evaluated. Different concentrations in a fixed ratio were added to the cultured cells and incubated for 48 h. Cell viability was evaluated using MTT and SRB assays. Synergism was evaluated using the Chou-Talalay method. The results indicate doxorubicin (DOX) and paclitaxel (PTX) alone at concentrations of their IC<sub>50</sub> and higher are cell growth inhibitors. Mefloquine, artesunate, and chloroquine at concentrations of their IC<sub>50</sub> demonstrate anti-cancer activity. In combination, almost all antimalarials demonstrate higher ability than DOX and PTX alone to decrease cell viability at concentrations of IC<sub>50</sub> and lower than their IC<sub>50</sub>. The combination of chloroquine, artesunate and mefloquine with DOX and PTX was synergic (CI < 1). The combination of DOX and mefloquine after 48 h incubation demonstrated the highest cytotoxicity against MCF-7 cells, and the combination of DOX and artesunate was the most synergic. These results suggest antimalarials could act synergistically with DOX/PTX for breast cancer therapy.
Project description:Two new series of synthetic renieramycins including 22-O-amino ester and hydroquinone 5-O-amino ester derivatives of renieramycin M were semi-synthesized and evaluated for their cytotoxicity against the metastatic non-small-cell lung cancer H292 and H460 cell lines. Interestingly, the series of 22-O-amino ester derivatives displayed a potent cytotoxic activity greater than the hydroquinone derivatives. The most cytotoxic derivative of the series was the 22-O-(N-Boc-l-glycine) ester of renieramycin M (5a: IC50 3.56 nM), which showed 7-fold higher potency than renieramycin M (IC50 24.56 nM) and 61-fold more than jorunnamycin A (IC50 217.43 nM) against H292 cells. In addition, 5a exhibited a significantly higher cytotoxic activity than doxorubicin (ca. 100 times). The new semi-synthetic renieramycin derivatives will be further studied and developed as potential cytotoxic agents for non-small-cell lung cancer treatment.
Project description:<b>Objective:</b> C49 is a chalcone derivative. The aim of the current study is to illuminate the efficacy of C49 in reversing multidrug resistance (MDR) in MCF-7/DOX cells and its underlying molecular mechanism. <b>Methods:</b> The cytotoxic effects of C49 on MCF-7/DOX cells were evaluated by MTT assay using different concentration (0-250 μmol/L) of C49. Cell proliferation was evaluated by colony formation assay. Cell death was examined by morphological analysis using Hoechst 33,258 staining. Flow cytometry and immunofluorescence were utilized to evaluate the intracellular accumulation of doxorubicin (DOX) and cell apoptosis. The differentially expressed genns between MCF-7 and MCF-7/DOX cells were analyzed by GEO database. The expression of PI3K/Akt pathway proteins were assessed by Western blot The activities of C49 combined with DOX was evaluated via xenograft tumor model in female BALB/c nude mice. <b>Results:</b> C49 inhibited the growth of MCF-7 cells (IC<sub>50</sub> = 59.82 ± 2.10 μmol/L) and MCF-7/DOX cells (IC<sub>50</sub> = 65.69 ± 8.11 μmol/L) with dosage-dependent and enhanced the cellular accumulation of DOX in MCF-7/DOX cells. The combination of C49 and DOX inhibited cell proliferation and promoted cell apoptosis. MCF-7/DOX cells regained drug sensibility with the combination treatment through inhibiting the expression of P-gp, p-PI3K and p-Akt proteins. Meanwhile, C49 significantly increased the anticancer efficacy of DOX <i>in vivo</i>. <b>Conclusion:</b> C49 combined with DOX restored DOX sensitivity in MCF-7/DOX cells through inhibiting P-gp protein.
Project description:Doxorubicin-based chemotherapy represents one of the most effective ways in combating human cancers. However, its clinical use is limited by severe side effects. Selenocystine (SeC) is a natural available selenoamino acid with novel anticancer efficacy. In this study, we used SeC to sensitize HepG2 human hepatocellular carcinoma (HCC) cells to DOX, and to achieve anticancer synergism in vitro and in vivo. Treatment with DOX dose-dependently reduced HepG2 cell viability through initiating cell apoptosis and strong G2/M phase cell cycle arrest. Mechanistic studies indicated that this sensitization of SeC to DOX was achieved by triggering inactivation of ERK and AKT and DNA damage through reactive oxygen species (ROS) overproduction. Pretreatment with inhibitors of ERK and AKT markedly enhanced combined treatment-induced cell killing, indicating that combined treatment-induced HCC cell killing with ERK- and AKT-dependent manner. Furthermore, inhibition of ROS effectively attenuated combined treatment-induced DNA damage and inactivation of ERK and AKT. Additionally, xenograft hepatocellular carcinoma growth was also effectively inhibited by combined treatment through induction of cell apoptosis in vivo. Taken together, our results suggest that the strategy to use SeC and DOX in combination could be a highly efficient way to achieve anticancer synergism against HCC.
Project description:Resistance to breast cancer (BCa) chemotherapy severely hampers the patient's prognosis. MicroRNAs provide a potential therapeutic prospect for BCa. In this study, the reversal function of microRNA34a (miR34a) on doxorubicin (Dox) resistance of BCa and the possible mechanism was investigated. We found that the relative level of miR34a was significantly decreased in Dox-resistant breast cancer cell MCF-7 (MCF-7/A) compared with Dox-sensitive MCF-7 cells. Transfection with miR34a significantly suppressed the invasion, migration, adhesion of MCF-7/A cells without inhibiting their growth obviously. The combination of miR34a and Dox could significantly inhibit the proliferation, migration, invasion and induce the apoptosis of MCF-7/A cells. The synergistic effect of this combination on resistant MCF-7/A cells has no obvious relation with the expressions of classical drug-resistant proteins P-GP, MRP and GST-<i>π</i>, while closely related with the down-regulation on TOP2A and BCRP. Moreover, we found both protein and mRNA expression of Snail were significantly up-regulated in MCF-7/A cells in comparison with MCF-7 cells. Transfection with small interfering RNA (siRNA) of Snail could inhibit the invasion, migration and adhesion of drug-resistant MCF-7/A cells, while high-expression of Snail could remarkably promote the invasion, migration and adhesion of MCF-7 cells, which might be related with regulation of N-cadherin and E-cadherin. Transfection with miR34a in MCF-7/A cells induced a decrease of Snail expression. The potential binding sites of miR34a with 3' UTR of Snail were predicted by miRDB target prediction software, which was confirmed by luciferase reporter gene method. Results showed that the relative activity of luciferase was reduced in MCF-7/A cells after co-transfection of miR34a and wild type (wt)-Snail, while did not change by co-transfection with miR34a and 3' UTR mutant type (mut) Snail. Combination of miR34a and Dox induced a stronger decrease of Snail in MCF-7/A cells in comparison to miR34a or Dox treatment alone. What' more, for the first time, we also found miR34a combined with Dox could obviously inhibit the expression of Snail through suppressing Notch/NF-<i>κ</i>B and RAS/RAF/MEK/ERK pathway in MCF-7/A cells. <i>In vivo</i> study indicated that combination of miR34a and Dox significantly slowed down tumor growth in MCF-7/A nude mouse xenograft model compared with Dox alone, which was manifested by the down-regulation of Snail and pro-apoptosis effect in tumor xenografts. These results together underline the relevance of miR34a-driven regulation of Snail in drug resistance and co-administration of miR34a and Dox may produce an effective therapy outcome in the future in clinic.
Project description:<h4>Background</h4>One key approach for anticancer therapy is drug combination. Drug combinations can help reduce doses and thereby decrease side effects. Furthermore, the likelihood of drug resistance is reduced. Distinct alterations in tumor metabolism have been described in past decades, but metabolism has yet to be targeted in clinical cancer therapy. Recently, we found evidence for synergism between dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and the HIF-1α inhibitor PX-478. In this study, we aimed to analyse this synergism in cell lines of different cancer types and to identify the underlying biochemical mechanisms.<h4>Methods</h4>The dose-dependent antiproliferative effects of the single drugs and their combination were assessed using SRB assays. FACS, Western blot and HPLC analyses were performed to investigate changes in reactive oxygen species levels, apoptosis and the cell cycle. Additionally, real-time metabolic analyses (Seahorse) were performed with DCA-treated MCF-7 cells.<h4>Results</h4>The combination of DCA and PX-478 produced synergistic effects in all eight cancer cell lines tested, including colorectal, lung, breast, cervical, liver and brain cancer. Reactive oxygen species generation and apoptosis played important roles in this synergism. Furthermore, cell proliferation was inhibited by the combination treatment.<h4>Conclusions</h4>Here, we found that these tumor metabolism-targeting compounds exhibited a potent synergism across all tested cancer cell lines. Thus, we highly recommend the combination of these two compounds for progression to in vivo translational and clinical trials.