The combination of doxorubicin and cyclophosphamide commonly used to treat breast cancer can cause premature ovarian failure and infertility. α–Tocopherol is a potent antioxidant whereas γ–tocopherol causes apoptosis in a variety of cancer models in vitro including breast cancer. We hypothesised that the combination of doxorubicin (Dox) and 4-hydroperoxycyclophosphamide (4-Cyc) would be more cytotoxic in vitro than each agent alone, and that α–tocopherol would reduce and γ–tocopherol would augment the cytotoxicity of the combined chemotherapeutics. Human MCF-7 breast cancer and KGN ovarian cells were exposed to Dox, 4-Cyc, combined Dox and 4-Cyc, α–tocopherol, γ–tocopherol, or a combination of Dox and 4-Cyc with α–tocopherol or γ–tocopherol. Cell viability was assessed using a crystal violet assay according to four schedules: 24h exposure, 24h exposure + 24h culture in medium, 24h exposure + 48h culture in medium, or 72h continuous exposure. Supernatants from each separate KGN culture experiment (n=3) were examined using an estradiol ELISA. Dox was cytotoxic to both MCF-7 and KGN cells, but 4-Cyc only killed MCF-7 cells. γ–Tocopherol significantly decreased MCF-7 but not KGN cell viability. The combined chemotherapeutics and γ–tocopherol were more cytotoxic to MCF-7 than KGN cells, and α–tocopherol reduced the cytotoxicity of the combined chemotherapeutics towards KGN ovarian cells, but not MCF-7 cells. The addition of both γ–tocopherol and α–tocopherol to the chemotherapeutic combination of Dox and cyclophosphamide has the potential to increase in vitro chemotherapeutic efficacy against breast cancer cells whilst decreasing cytotoxicity towards ovarian granulosa cells.

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Multi-drug resistance (MDR) presents a serious problem in cancer chemotherapy. In this study, Vitamin E (VE)-Albumin core-shell nanoparticles were developed for paclitaxel (PTX) delivery to improve the chemotherapy efficacy in an MDR breast cancer model. The PTX-loaded VE-Albumin core-shell nanoparticles (PTX-VE NPs) had small particle sizes (about 100 nm), high drug entrapment efficiency (95.7%) and loading capacity (12.5%), and showed sustained release profiles, in vitro. Docking studies indicated that the hydrophobic interaction and hydrogen bonds play a significant role in the formation of the PTX-VE NPs. The results of confocal laser scanning microscopy analysis demonstrated that the cell uptake of PTX was significantly increased by the PTX-VE NPs, compared with the NPs without VE (PTX NPs). The PTX-VE NPs also exhibited stronger cytotoxicity, compared with PTX NPs with an increased accumulation of PTX in the MCF-7/ADR cells. Importantly, the PTX-VE NPs showed a higher anti-cancer efficacy in MCF-7/ADR tumor xenograft model than the PTX NPs and the PTX solutions. Overall, the VE-Albumin core-shell nanoparticles could be a promising nanocarrier for PTX delivery to improve the chemotherapeutic efficacy of MDR cancer.

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Tocopherols and tocotrienols have been extensively studied due to their anticancer potential, especially against breast cancer. Therefore, the aim of this study was to quantitatively determine tocochromanols in human breast adipose tissue with the use of HPLC-FLD. The sample preparation procedure included homogenization and solvent extraction with isopropanol/ethanol/0.1% formic acid mixture prior to solid phase extraction (SPE). After implementation of central composite design (CCD), satisfactory separation of all eight target compounds was achieved within 10.5 min. Chromatographic runs were carried out with the use of a naphthylethyl chromatographic column with methanol/water mixture (89/11, v/v) as the mobile phase. Fluorescence detection of tocochromanols was performed with excitation and emission wavelengths 298 and 330 nm, respectively. The method was validated in terms of linearity, carryover, recovery, precision, accuracy and stability. Extraction yield was also determined for accurate evaluation of vitamin E content in human breast adipose tissue samples. Finally, concentrations of particular tocochromanols compounds were assessed in human breast adipose tissue samples obtained from 99 patients, including women with breast cancer, healthy volunteers and women deceased by accident. The raw data was transformed according to the newly developed equation for accurate estimation of tocochromanols’ concentrations in breast adipose tissue samples. Results obtained in the study indicated that proposed analytical assay could be useful in breast cancer research.

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OBJECTIVE:

To assess the role of Vitamin E to improve the survival of Wharton’s jelly derived mesenchymal stem cells (WJMSCs) in breast cancer conditions.

STUDY DESIGN:

An experimental study.

PLACE AND DURATION OF STUDY:

Centre for Research in Molecular Medicine, University of Lahore, from November 2016 to March 2017.

METHODOLOGY:

WJMSCs were obtained from umbilical cord tissue with enzyme digestion method. Isolated cells were characterized for CD90 and CD45 by immunocytochemistry. Pretreatment and conjugation therapies of vitamin E in 50mM and 100mM concentration were used on WJMSCs and breast cancer plasma was provided to mimic the cancer conditions, while WJMSCs provided with normal plasma were considered control. Cells’ viability, proliferation and death were evaluated by crystal violet staining, MTT assay and LDH assay, respectively. Oxidative stress was observed by activity of anti-oxidant enzymes (GSH, catalase, SOD) and reactive oxygen species (MDA).

RESULTS:

The isolated cells expressed mesenchymal stem cells marker CD90 and lacked hematopoietic marker CD45. Vitamin E improved the viability and proliferation of WJMSCs in normal plasma, in conjugation with breast cancer plasma and in pretreatment groups but conjugation group showed even better results with concentration of 100mM as compared to the pretreatment group and opposite was observed for LDH assay for cells death analysis. Vitamin E also reduced the oxidative stress in 100mM more pronounced in conjugation group as compared to pretreatment group while left no harmful effects on WJMSCs in normal plasma.

CONCLUSION:

Vitamin E conjugation with breast cancer conditions significantly improved growth of WJMSCs. Thus vitamin E treated WJMSCs are better therapeutic options for breast cancer.

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AIM:

To develop 6-O-palmitoyl-ascorbic acid-based niosomes targeted to transferrin receptor for intravenous administration of tocotrienols(T3) in breast cancer.

MATERIALS & METHODS:

Niosomes were prepared using film hydration and ultrasonication methods. Transferrin was coupled to the surface of niosomes via chemical linker. Nanovesicles were characterized for size, zeta potential, morphology, stability and biological efficacy.

RESULTS:

When evaluated in MDA-MB-231 cells, entrapment of T3 in niosomes caused 1.5-fold reduction in IC₅₀ value compared with nonformulated T3. In vivo, the average tumor volume of mice treated with tumor-targeted niosomes was 12-fold lower than that of untreated group, accompanied by marked downregulation of three genes involved in metastasis.

CONCLUSION:

Findings suggested that tumor-targeted niosomes served as promising delivery system for T3 in cancer therapy.

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Overexpression of heregulin, a potent ligand that activates HER3 and HER4 receptors, plays a significant role in the development of chemotherapy resistance in breast cancer patients. Exosomes released from cancer cells are small vesicles originating from the outward budding of lipid rafts that carry various mitogenic proteins that then act locally in an autocrine/paracrine manner to stimulate cancer cell growth. Since the anticancer activity of γ-tocotrienol has been shown to be mediated in part through the disruption of lipid rafts, studies were conducted to determine the effect of γ-tocotrienol on exosomes mitogenic biopotency. Exosomes isolated from the media of cultured T47D breast cancer cells were found to stimulate T47D cell growth in a dose-dependent manner. These growth stimulating effects were due to the high levels of heregulin contained in the exosomes that act to stimulate HER3 and HER4 activation, heterodimerization and mitogenic signaling. Exposure to 5 μM γ-tocotrienol resulted in the selective accumulation and disruption in the integrity of the lipid raft microdomain and a corresponding decrease in exosome heregulin content and mitogenic biopotency. These findings provide strong evidence indicating that the anticancer effects of γ-tocotrienol are mediated, at least in part, by directly disrupting HER dimerization and signaling within the lipid rafts and indirectly by reducing exosome heregulin content and subsequent autocrine/paracrine mitogenic stimulation.

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Estrogens have been implicated as complete carcinogens for breast and other tissues through mechanisms involving increased cell proliferation, oxidative stress, and DNA damage. Because of their potent antioxidant activity and other effects, tocopherols have been shown to exert antitumor activities in various cancers. However, limited information is available on the effect of different forms of tocopherols in estrogen-mediated breast cancer. To address this, we examined the effects of α-, γ-, and δ-tocopherols as well as a natural γ-tocopherol-rich mixture of tocopherols, γ-TmT, on estrogen-stimulated MCF-7 cells in vitro and in vivo For the in vivo studies, MCF-7 cells were injected into the mammary fat pad of immunodeficient mice previously implanted with estrogen pellets. Mice were then administered diets containing 0.2% α-, γ-, δ-tocopherol, or γ-TmT for 5 weeks. Treatment with α-, γ-, δ-tocopherols, and γ-TmT reduced tumor volumes by 29% (P < 0.05), 45% (P < 0.05), 41% (P < 0.05), and 58% (P < 0.01), as well as tumor weights by 20%, 37% (P < 0.05), 39% (P < 0.05), and 52% (P < 0.05), respectively. γ- and δ-tocopherols and γ-TmT inhibited the expression of cell proliferation-related genes such as cyclin D1 and c-Myc, and estrogen-related genes such as TFF/pS2, cathepsin D, and progesterone receptor in estrogen-stimulated MCF-7 cells in vitro Further, γ- and δ-tocopherols decreased the levels of estrogen-induced oxidative stress and nitrosative stress markers, 8-hydroxy-2′-deoxyguanosine and nitrotyrosine, as well as the DNA damage marker, γ-H2AX. Our results suggest that γ- and δ-tocopherols and the γ-tocopherol-rich mixture are effective natural agents for the prevention and treatment of estrogen-mediated breast cancer.

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In addition to its antioxidant properties, γ-tocotrienol also has the ability to inhibit HMG-CoA reductase, which is the key enzyme in the mevalonate pathway for cholesterol biosynthesis. Statins, the competitive inhibitors of HMG-CoA reductase, display potent anticancer activity and reversal ability of multidrug resistance in a variety of tumor cells, which is believed to be due to their inhibition of HMG-CoA reductase. Here, we determined the role of the mevalonate pathway in γ-tocotrienol-mediated reversal of multidrug resistance in cancer cells. We found both γ-tocotrienol and atorvastatin effectively reversed multidrug resistance of MCF-7/Adr and markedly inhibited the intracellular levels of FPP and GGPP. Exogenous addition of mevalonate or FPP and GGPP almost completely prevented the reversal ability of atorvastatin but only partly attenuated the reversal effect of γ-tocotrienol on doxorubicin resistance. In addition, γ-tocotrienol actively inhibited the expression of P-gp and increased the accumulation of doxorubicin in cells, which led to the enhanced G2/M arrest and cell apoptosis. Taken together, γ-tocotrienol reversed the multidrug resistance of MCF-7/Adr with a mechanism distinct from that of atorvastatin. Instead of the mevalonate pathway, the inhibition of P-gp expression is a potential mechanism by which γ-tocotrienol reverses multidrug resistance in MCF-7/Adr.

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