Tuning mPEG-PLA/vitamin E-TPGS-based Mixed Micelles for Combined Celecoxib/Honokiol Therapy for Breast Cancer

Jiahui Sun, Jing Li, Qi Liu, Min Jiang, Mengjia Yang, Siwen Zhan, Tong Qiu, Kaiyong He, Xueqiong Zhang

Eur J Pharm Sci . 2020 Apr 15;146:105277. doi: 10.1016/j.ejps.2020.105277. Epub 2020 Feb 24.

Abstract

This study aimed to develop, evaluate, and optimize the mPEG-PLA/vitamin E-TPGS mixed micelle drug delivery system to encapsulate celecoxib (CXB) and honokiol (HNK) for intravenous treatment of breast cancer. To this end, we formulated CXB-loaded mPEG-PLA/vitamin E-TPGS (PV-CXB) and HNK-loaded mPEG-PLA/vitamin E-TPGS (PV-HNK) mixed micelles and analyzed their characteristics. The 4T1 cell line was used for cytotoxicity determination and cellular uptake experiments, and for establishing a 4T1-bearing mouse model for histopathology, immunofluorescence, terminal deoxynucleotidyl transferase-mediated nick end labeling, and Western blot analysis. The synergistic effects of PV-CXB and PV-HNK combination therapy were investigated in vitro and in vivo using the coefficient of drug interaction values. The mean size of PV-CXB and PV-HNK prepared with optimal formulation was approximately 50 nm, with a spherical shape. PV-CXB and PV-HNK combination therapy exhibited cytotoxicity in 4T1 cells in vitro. However, the toxicity of PV-CXB and PV-HNK combination therapy was not apparent in normal tissues (heart, liver, spleen, lung, and kidney) in vivo and reduced the expression of collagen fibers in tumor tissues. Moreover, the combination therapy reduced the expression of tumor growth biomarkers (Foxp3, CD4, Gr-1, CD11b, CD31, Ki67, FoxM1, and VEGF). In addition, the tumor cell apoptosis rate reached 45.71 ± 0.62%. The combined treatment with PV-CXB and PV-HNK showed synergistic effect both in vitro and in vivo. Thus, the PV-CXB and PV-HNK drug delivery system could be used as a potential combination therapy for breast cancer .

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Beta-Tocotrienol Exhibits More Cytotoxic Effects than Gamma-Tocotrienol on Breast Cancer Cells by Promoting Apoptosis via a P53-Independent PI3-Kinase Dependent Pathway

Idriss M, Hodroj MH, Fakhoury R, Rizk S

Biomolecules. 2020 Apr 9;10(4). pii: E577. doi: 10.3390/biom10040577.

Abstract

Studies on tocotrienols have progressively revealed the benefits of these vitamin E isoforms on human health. Beta-tocotrienol (beta-T3) is known to be less available in nature compared to other vitamin E members, which may explain the restricted number of studies on beta-T3. In the present study, we aim to investigate the anti-proliferative effects and the pro-apoptotic mechanisms of beta-T3 on two human breast adenocarcinoma cell lines MDA-MB-231 and MCF7. To assess cell viability, both cell lines were incubated for 24 and 48 h, with different concentrations of beta-T3 and gamma-T3, the latter being a widely studied vitamin E isoform with potent anti-cancerous properties. Cell cycle progression and apoptosis induction upon treatment with various concentrations of the beta-T3 isoform were assessed. The effect of beta-T3 on the expression level of several apoptosis-related proteins p53, cytochrome C, cleaved-PARP-1, Bax, Bcl-2, and caspase-3, in addition to key cell survival proteins p-PI3K and p-GSK-3 α/β was determined using western blot analysis. Beta-tocotrienol exhibited a significantly more potent anti-proliferative effect than gamma-tocotrienol on both cell lines regardless of their hormonal receptor status. Beta-T3 induced a mild G1 arrest on both cell lines, and triggered a mitochondrial stress-mediated apoptotic response in MDA-MB-231 cells. Mechanistically, beta-T3’s anti-neoplastic activity involved the downregulation of phosphorylated PI3K and GSK-3 cell survival proteins. These findings suggest that vitamin E beta-T3 should be considered as a promising anti-cancer agent, more effective than gamma-T3 for treating human breast cancer and deserves to be further studied to investigate its effects in vitro and on other cancer types.

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Enhanced Prevention of Breast Tumor Metastasis by Nanoparticle-Delivered Vitamin E in Combination with Interferon-Gamma

Wu Y, Liu J, Movahedi F, Gu W, Xu T, Xu ZP

Adv Healthc Mater. 2020 Feb 13:e1901706. doi: 10.1002/adhm.201901706. [Epub ahead of print]

Abstract

Preventing cancer metastasis is one of the remaining challenges in cancer therapy. As an efficient natural product, alpha-tocopheryl succinate (α-TOS), the most effective form of vitamin E, holds great anticancer potential. To improve its efficacy and bioavailability, lipid-coated calcium carbonate/phosphate (LCCP) nanoparticles (NPs) with folic acid and PEG modification are synthesized for efficient delivery of α-TOS to 4T1 cancer cells. The optimized LCCP-FA NPs (NP-TOS15) show an α-TOS loading efficiency of around 60%, and enhanced uptake by 4T1 metastatic cancer cells. Consequently, NP-TOS15 significantly enhance the anticancer effect in combination with interferon-gamma (IFN-γ) in terms of apoptosis facilitation and migration inhibition. Importantly, NP-TOS15 upregulate the anticancer immunity via downregulating program death ligand 1 (PD-L1) expression that is initially induced by IFN-γ, and remarkably prevent the lung metastasis, particularly in combination with IFN-γ. Further investigation reveals that this combination therapy also modulates the cytotoxic lymphocyte infiltration into the tumor microenvironment for tumor elimination. Taken together, the NP delivery of α-TOS in combination with IFN-γ provides an applicable strategy for cancer therapy.

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Advancing the Role of Gamma-Tocotrienol as Proteasomes Inhibitor: A Quantitative Proteomic Analysis of MDA-MB-231 Human Breast Cancer Cells

Ramdas P, Radhakrishnan AK, Abdu Sani AA, Kumari M, Anandha Rao JS, Abdul-Rahman PS

Biomolecules. 2019 Dec 21;10(1). pii: E19. doi: 10.3390/biom10010019.

Abstract

Tocotrienol, an analogue of vitamin E has been known for its numerous health benefits and anti-cancer effects. Of the four isoforms of tocotrienols, gamma-tocotrienol (γT3) has been frequently reported for their superior anti-tumorigenic activity in both in vitro and in vivo studies, when compared to its counterparts. In this study, the effect of γT3 treatment in the cytoplasmic and nuclear fraction of MDA-MB-231 human breast cancer cells were assessed using the label-free quantitative proteomics analysis. The cytoplasmic proteome results revealed the ability of γT3 to inhibit a group of proteasome proteins such as PSMA, PSMB, PSMD, and PSME. The inhibition of proteasome proteins is known to induce apoptosis in cancer cells. As such, the findings from this study suggest γT3 as a potential proteasome inhibitor that can overcome deficiencies in growth-inhibitory or pro-apoptotic molecules in breast cancer cells. The nuclear proteome results revealed the involvement of important nuclear protein complexes which hardwire the anti-tumorigenesis mechanism in breast cancer following γT3 treatment. In conclusion, this study uncovered the advancing roles of γT3 as potential proteasomes inhibitor that can be used for the treatment of breast cancer.

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γ-Tocotrienol Suppression of the Warburg Effect Is Mediated by AMPK Activation in Human Breast Cancer Cells

Dronamraju V, Ibrahim BA, Briski KP, Sylvester PW

Nutr Cancer. 2019;71(7):1214-1228. doi: 10.1080/01635581.2019.1599969.

Abstract

Cancer cell metabolism is characterized by aerobic glycolysis or the “Warburg effect”. Enhanced Akt signaling is associated with activation of various downstream enzymes involved in the glycolytic process, whereas activation of 5′-AMP-activated kinase (AMPK) acts to terminate energy expending mechanisms and decrease glycolytic enzyme expression. Studies were conducted to determine if the anticancer effects of γ-tocotrienol, are mediated through a suppression in aerobic glycolysis. Results show that treatment with 0-7 μM γ-tocotrienol throughout a 4-day culture period resulted in a dose-responsive increase in AMPK activation, and corresponding decrease in Akt activity in human MCF-7 and MDA-MB-231 breast cancer cells. γ-Tocotrienol treatment was also found to induce a dose-responsive decrease in phosphorylated-Fox03 (inactivated), a transcription factor that acts to inhibit in the levels of glycolytic enzyme, and this decrease was associated with a reduction in glycolytic enzyme levels and activity, as well as glucose consumption in these cells. PCR microarray analysis shows that γ-tocotrienol treatment decreases the expression of genes associate with metabolic signaling and glycolysis in MCF-7 and MDA-MB-231 breast cancer cells. In summary, these findings demonstrate that the anticancer effects of γ-tocotrienol are mediated, at least in part, by a suppression in the Warburg effect.

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Tocotrienols Modulate Breast Cancer Secretomes and Affect Cancer-Signaling Pathways in MDA-MB-231 Cells: A Label-Free Quantitative Proteomic Analysis.

Ramdas P, Radhakrishnan AK, Abdu Sani AA, Abdul-Rahman PS

Nutr Cancer. 2019;71(8):1263-1271. doi: 10.1080/01635581.2019.1607407

Abstract

Tocotrienols (T3), a family of vitamin E, are reported to possess potent anti-cancer effects but the molecular mechanisms behind these effects still remain unclear. The aim of this study was to investigate how T3 exert anti-cancer effects on MDA-MB-231 human breast cancer cells. The MDA-MB-231 cells were chosen for this study as they are triple-negative and highly metastatic cells, which form aggressive tumors in experimental models. The MDA-MB-231 cells were treated with varying concentrations (0-20 µg mL-1) of gamma (γ) or delta (δ) T3 and the secretome profiles of these cells treated with half maximal inhibitory concentration (IC50) of γT3 (5.8 µg mL-1) or δT3 (4.0 µg mL-1) were determined using label-free quantitative proteomic strategy. A total of 103, 174 and 141 proteins were identified with ProteinLynx Global Server (PLGS) score of more than 200 and above 25% sequence coverage in the untreated control and T3-treated cell culture supernatant respectively. A total of 18 proteins were dysregulated between untreated control and T3 (δT3 or γT3) treated conditions. The results showed that T3 treatment downregulated the exogenous Cathepsin D and Serpine1 proteins but upregulated Profilin-1 protein, which play a key role in breast cancer in the MDA-MB-231 cells. These findings strongly suggest that T3 may induce differential expression of secreted proteins involved in the cytoskeletal regulation of RHO GTPase signaling pathway.

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Palm Tocotrienol-Adjuvanted Dendritic Cells Decrease Expression of the SATB1 Gene in Murine Breast Cancer Cells and Tissues

Abdul Hafid SR, Radhakrishnan AK

Vaccines (Basel). 2019 Nov 27;7(4). pii: E198. doi: 10.3390/vaccines7040198.

Abstract

The aim of this study was to evaluate the effectiveness of immunotherapy using dendritic cells (DC) pulsed with tumor lysate (a DC vaccine) in combination with daily supplementation of tocotrienol-rich fraction (TRF) to potentiate anti-tumor immune responses. We had previously reported that DC-vaccine immunotherapy together with TRF supplementation induced protective immunity to tumor challenge. Breast cancer was induced in female BALB/c mice. The mice were randomly assigned into the treatment groups. At autopsy, peripheral blood was collected in heparinized tube and the expression of cell surface molecules (CD40, CD80, CD83, and CD86) that are crucial for T-cell activation and survival were analyzed by flow cytometry. Tumor was excised from each animal and snap-frozen. Total RNA was extracted from each tumor tissue for microarray and gene expression analysis. Total protein was extracted from tumor tissue for protein expression studies using Western blotting. The results show that systemic administration of 1 mg TRF daily in combination with DC-vaccine immunotherapy (DC + TL + TRF) caused a marked reduction (p < 0.05) of tumor size and increased (p < 0.05) the survival rates of the tumor-inoculated mice. The expression of CD40, CD80, CD83, and CD86 were upregulated in peripheral blood from the DC + TL + TRF group compared to other groups. In addition, there was higher expression of FasL in tumor-excised mice from the DC + TL + TRF group compared to other groups. FasL plays an important role in maintaining immune privilege and is required for cytotoxic T-lymphocyte (CTL) activity. Microarray analysis identified several genes involved in the regulation of cancer. In this study, we focused on the special AT rich binding protein 1 (SATB1) gene, which was reported to have dual functions, one of which was to induce aggressive growth in breast cancer cells. Tumors from DC + TL + TRF mice showed lower (p < 0.05) expression of SATB1 gene. Further study will be conducted to investigate the molecular functions of and the role of SATB1 in 4T1 mammary cancer cells and DC. In conclusion, TRF supplementation can potentiate the effectiveness of DC-vaccine immunotherapy.

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Palm Tocotrienol-Adjuvanted Dendritic Cells Decrease Expression of the SATB1 Gene in Murine Breast Cancer Cells and Tissues

Abdul Hafid SR, Radhakrishnan AK

Vaccines (Basel). 2019 Nov 27;7(4). pii: E198. doi: 10.3390/vaccines7040198.

Abstract

The aim of this study was to evaluate the effectiveness of immunotherapy using dendritic cells (DC) pulsed with tumor lysate (a DC vaccine) in combination with daily supplementation of tocotrienol-rich fraction (TRF) to potentiate anti-tumor immune responses. We had previously reported that DC-vaccine immunotherapy together with TRF supplementation induced protective immunity to tumor challenge. Breast cancer was induced in female BALB/c mice. The mice were randomly assigned into the treatment groups. At autopsy, peripheral blood was collected in heparinized tube and the expression of cell surface molecules (CD40, CD80, CD83, and CD86) that are crucial for T-cell activation and survival were analyzed by flow cytometry. Tumor was excised from each animal and snap-frozen. Total RNA was extracted from each tumor tissue for microarray and gene expression analysis. Total protein was extracted from tumor tissue for protein expression studies using Western blotting. The results show that systemic administration of 1 mg TRF daily in combination with DC-vaccine immunotherapy (DC + TL + TRF) caused a marked reduction (p < 0.05) of tumor size and increased (p < 0.05) the survival rates of the tumor-inoculated mice. The expression of CD40, CD80, CD83, and CD86 were upregulated in peripheral blood from the DC + TL + TRF group compared to other groups. In addition, there was higher expression of FasL in tumor-excised mice from the DC + TL + TRF group compared to other groups. FasL plays an important role in maintaining immune privilege and is required for cytotoxic T-lymphocyte (CTL) activity. Microarray analysis identified several genes involved in the regulation of cancer. In this study, we focused on the special AT rich binding protein 1 (SATB1) gene, which was reported to have dual functions, one of which was to induce aggressive growth in breast cancer cells. Tumors from DC + TL + TRF mice showed lower (p < 0.05) expression of SATB1 gene. Further study will be conducted to investigate the molecular functions of and the role of SATB1 in 4T1 mammary cancer cells and DC. In conclusion, TRF supplementation can potentiate the effectiveness of DC-vaccine immunotherapy.

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Vitamin E D-alpha-tocopheryl polyethylene glycol 1000 succinate-conjugated liposomal docetaxel reverses multidrug resistance in breast cancer cells

Li N, Fu T, Fei W, Han T, Gu X, Hou Y, Liu Y, Yang J

J Pharm Pharmacol. 2019 Aug;71(8):1243-1254. doi: 10.1111/jphp.13126. Epub 2019 Jun 18.

Abstract

OBJECTIVES:

Multidrug resistance (MDR) remains a primary challenge in breast cancer treatment. In the present study, D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS)-coated docetaxel-loaded liposomes were developed as a novel drug delivery system to reverse MDR and enhance breast cancer therapy compared with the traditional liposomes, DSPE-mPEG-coated liposomes (stealth liposomes) and commercial Taxotere® .

KEY FINDINGS:

Liposomes were prepared by thin – film dispersion method. Evaluations were performed using human breast cancer MCF-7 and resistant MCF-7/ADR cells. The reversal multidrug-resistant effect was assessed by P-gp inhibition assay, cytotoxicity, cellular uptake and apoptosis assay.

RESULTS:

The TPGS-chol-liposomes were of an appropriate particle size (140.0 ± 6.0 nm), zeta potential (-0.196 ± 0.08 mv), high encapsulation efficiency (99.0 ± 0.9) and favourable in vitro sustained release. The TPGS-coated liposomes significantly improved cytotoxicity and increased the intracellular accumulation of docetaxel in both types of breast cancer cells. The TPGS-coated liposomes were confirmed to induce apoptosis via a synergistic effect between docetaxel and TPGS. It was demonstrated that TPGS enhanced the intracellular accumulation of drug by inhibiting overexpressed P-glycoprotein.

CONCLUSIONS:

The TPGS-conjugated liposomes showed significant advantages in vitro compared with the PEG-conjugated liposomes. The TPGS-conjugated liposomes could reverse the MDR and enhance breast cancer therapy.

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Tocotrienols Modulate Breast Cancer Secretomes and Affect Cancer-Signaling Pathways in MDA-MB-231 Cells: A Label-Free Quantitative Proteomic Analysis

Ramdas P, Radhakrishnan AK, Abdu Sani AA, Abdul-Rahman PS

Nutr Cancer. 2019 May 14:1-9. doi: 10.1080/01635581.2019.1607407. [Epub ahead of print]

Abstract

Tocotrienols (T3), a family of vitamin E, are reported to possess potent anti-cancer effects but the molecular mechanisms behind these effects still remain unclear. The aim of this study was to investigate how T3 exert anti-cancer effects on MDA-MB-231 human breast cancer cells. The MDA-MB-231 cells were chosen for this study as they are triple-negative and highly metastatic cells, which form aggressive tumors in experimental models. The MDA-MB-231 cells were treated with varying concentrations (0-20 µg mL-1) of gamma (γ) or delta (δ) T3 and the secretome profiles of these cells treated with half maximal inhibitory concentration (IC50) of γT3 (5.8 µg mL-1) or δT3 (4.0 µg mL-1) were determined using label-free quantitative proteomic strategy. A total of 103, 174 and 141 proteins were identified with ProteinLynx Global Server (PLGS) score of more than 200 and above 25% sequence coverage in the untreated control and T3-treated cell culture supernatant respectively. A total of 18 proteins were dysregulated between untreated control and T3 (δT3 or γT3) treated conditions. The results showed that T3 treatment downregulated the exogenous Cathepsin D and Serpine1 proteins but upregulated Profilin-1 protein, which play a key role in breast cancer in the MDA-MB-231 cells. These findings strongly suggest that T3 may induce differential expression of secreted proteins involved in the cytoskeletal regulation of RHO GTPase signaling pathway.

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