Engineering Nanoplatform for Combined Cancer Therapeutics via Complementary Autophagy Inhibition.

Despite advances in the development of tumor treatments, mortality from cancer continues to increase. Nanotechnology is expected to provide an innovative anti-cancer therapy, to combat challenges such as multidrug resistance and tumor recurrence. Nevertheless, tumors can greatly rely on autophagy as an alternative source for metabolites, and which desensitizes cancer cells to therapeutic stress, hindering the success of any current treatment paradigm. Autophagy is a conserved process by which cells turn over their own constituents to maintain cellular homeostasis. The multistep autophagic pathway provides potentially druggable targets to inhibit pro-survival autophagy under various therapeutic stimuli. In this review, we focus on autophagy inhibition based on functional nanoplatforms, which may be a potential strategy to increase therapeutic sensitivity in combinational cancer therapies, including chemotherapy, radiotherapy, phototherapy, sonodynamic therapy, and immunotherapy.

Ionizing radiation downregulates estradiol synthesis via endoplasmic reticulum stress and inhibits the proliferation of estrogen receptor-positive breast cancer cells.

Breast cancer is a major threat to women's health and estrogen receptor-positive (ER+) breast cancer exhibits the highest incidence among these cancers. As the primary estrogen, estradiol strongly promotes cellular proliferation and radiotherapy, as a standard treatment, exerts an excellent therapeutic effect on ER+ breast cancer. Therefore, we herein wished to explore the mechanism(s) underlying the inhibitory effects of radiation on the proliferation of ER+ breast cancer cells. We used the ER+ breast cancer cell lines MCF7 and T47D, and their complementary tamoxifen-resistant cell lines in our study. The aforementioned cells were irradiated at different doses of X-rays with or without exogenous estradiol. CCK8 and clone-formation assays were used to detect cellular proliferation, enzyme-linked immunosorbent assay (ELISA) to determine estradiol secretion, western immunoblotting analysis and quantitative real-time PCR to evaluate the expression of proteins, and immunofluorescence to track endoplasmic reticulum stress-related processes. Finally, BALB/C tumor-bearing nude mice were irradiated with X-rays to explore the protein expression in tumors using immunohistochemistry. We found that ionizing radiation significantly reduced the phosphorylation of estrogen receptors and the secretion of estradiol by ER+ breast cancer cells. CYP19A (aromatase) is an enzyme located in the endoplasmic reticulum, which plays a critical role in estradiol synthesis (aromatization), and we further demonstrated that ionizing radiation could induce endoplasmic reticulum stress with or without exogenous estradiol supplementation, and that it downregulated the expression of CYP19A through ER-phagy. In addition, ionizing radiation also promoted lysosomal degradation of CYP19A, reduced estradiol synthesis, and inhibited the proliferation of tamoxifen-resistant ER+ breast cancer cells. We concluded that ionizing radiation downregulated the expression of CYP19A and reduced estradiol synthesis by inducing endoplasmic reticulum stress in ER+ breast cancer cells, thereby ultimately inhibiting cellular proliferation.

Pulsed and Discontinuous Electromagnetic Field Exposure Decreases Temozolomide Resistance in Glioblastoma by Modulating the Expression of O6-Methylguanine-DNA Methyltransferase, Cyclin-D1, and p53.

Background: Glioblastoma is a malignant and very aggressive brain tumor with a poor prognosis. Despite having chemotherapy concomitant with surgery and/or radiation therapy, the median survival of glioblastoma-affected people is less than 1 year. Temozolomide (TMZ) is a chemotherapeutic used as a first line treatment of glioblastoma. Several studies have reported that resistance to TMZ due to overexpression of O6-methylguanine-DNA methyltransferase (MGMT) is the main reason for treatment failure. Several studies described that pulsed-electromagnetic field (EMF) exposure could induce cell death and influence gene expression. Materials and Methods: In this study the authors assessed the effects of EMF (50 Hz, 70 G) on cytotoxicity, cell migration, gene expression, and protein levels in TMZ-treated T98 and A172 cell lines. Results: In this study, the authors show that treatment with a combination of TMZ and EMF enhanced cell death and decreased the migration potential of T98 and A172 cells. The authors also observed overexpression of the p53 gene and downregulation of cyclin-D1 protein in comparison to controls. In addition, T98 cells expressed the MGMT protein following treatment, while the A172 cells did not express MGMT. Conclusion: Their data indicate that EMF exposure improved the cytotoxicity of TMZ on T98 and A172 cells and could partially affect resistance to TMZ in T98 cells.

Microwave hyperthermia enhances the sensitivity of lung cancer cells to gemcitabine through reactive oxygen species­induced autophagic death.

The pleiotropic effects of hyperthermia on cancer cells have been well documented, and microwave hyperthermia (MWHT) has been widely applied for multifarious cancer treatment. However, the mechanisms underlying the anticancer effect of MWHT combined with gemcitabine (GEM) remain poorly understood. The aim of the present study was to investigate the role of autophagy in the thermo­chemotherapy of human squamous cell lung carcinoma cells. It was observed that MWHT combined with GEM potently suppressed the viability of NCI­H2170 and NCI­H1703 cells, and induced G0/G1 cell cycle arrest. Notably, MWHT with GEM induced autophagy, as indicated by the formation of autophagic vacuoles, downregulation of p62 and upregulation of light chain 3­II. It was further demonstrated that the autophagy was due to the production of reactive oxygen species (ROS), whereas N­acetyl cysteine, an ROS scavenger, attenuated the level of autophagy. However, when the autophagy inhibitor 3­methyladenine was used, there was no significant change in the production of ROS. Furthermore, it was observed that MWHT combined with GEM downregulated the protein expression levels of phosphoinositide 3­kinase (PI3K), phosphorylated (p)­PI3K, protein kinase B (AKT), p­AKT, mammalian target of rapamycin (mTOR), p­mTOR, phosphorylated S6 (pS6) and p70 S6 kinase, which are associated with autophagy. In addition, the results demonstrated that ROS served as an upstream mediator of PI3K/AKT/mTOR signaling. In light of these findings, the present study provides original insights into the molecular mechanisms underlying the cell death induced by MWHT combined with GEM, and this may be a promising approach for the treatment of human squamous cell lung carcinoma.

The Static Magnetic Field Remotely Boosts the Efficiency of Doxorubicin through Modulating ROS Behaviors.

Exposure to magnetic field (MF) can affect cellular metabolism remotely. Cardio-toxic effects of Doxorubicin (DOXO) have limited clinical uses at high dose. MF due to its effect on reactive oxygen species (ROS) lifetime, may provide a suitable choice to boost the efficacy of this drug at low dose. Here, we investigated the potential effects of homogenous static magnetic field (SMF) on DOXO-induced toxicity and proliferation rate of cancer cells. The results indicated that SMF similar to DOXO decreased the cell viability as well as the proliferation rate of MCF-7 and HFF cells. Moreover, combination of 10 mT SMF and 0.1 µM DOXO decreased the viability and proliferation rate of cancer and normal cells in a synergetic manner. In spite of high a GSH level in cancer cell, SMF boosts the generation and lifetime of ROS at low dose of DOXO, and overcame to GSH mediated drug resistance. The results also confirmed that SMF exposure decreased 50% iron content of cells, which is attributed to iron homeostasis. In conclusion, these findings suggest that SMF can decrease required dose of chemotherapy drugs such as DOXO and thereby decrease their side effect.

The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells.

Modulation of the redox system in cancer cells has been considered a promising target for anti-cancer therapy. The novel MTH1 inhibitor TH588 proved tremendous potential in terms of cancer cell eradication, yet its specificity has been questioned by recent reports, indicating that TH588 may also induce cancer cell death by alternative mechanisms than MTH1 inhibition. Here we used a panel of heterogeneous neuroendocrine tumor cells in order to assess cellular mechanisms and molecular signaling pathways implicated in the effects of TH588 alone as well as dual-targeting approaches combining TH588 with everolimus, cytotoxic 5-fluorouracil or γ-irradiation. Our results reflect that TH588 alone efficiently decreased the survival of neuroendocrine cancer cells by PI3K-Akt-mTOR axis downregulation, increased apoptosis and oxidative stress. However, in the dual-targeting approaches cell survival was further decreased due to an even stronger downregulation of the PI3K-Akt-mTOR axis and augmentation of apoptosis but not oxidative stress. Furthermore, we could attribute TH588 chemo- and radio-sensitizing properties. Collectively our data not only provide insights into how TH588 exactly kills cancer cells but also depict novel perspectives for combinatorial treatment approaches encompassing TH588.

BEMER Electromagnetic Field Therapy Reduces Cancer Cell Radioresistance by Enhanced ROS Formation and Induced DNA Damage.

Each year more than 450,000 Germans are expected to be diagnosed with cancer subsequently receiving standard multimodal therapies including surgery, chemotherapy and radiotherapy. On top, molecular-targeted agents are increasingly administered. Owing to intrinsic and acquired resistance to these therapeutic approaches, both the better molecular understanding of tumor biology and the consideration of alternative and complementary therapeutic support are warranted and open up broader and novel possibilities for therapy personalization. Particularly the latter is underpinned by the increasing utilization of non-invasive complementary and alternative medicine by the population. One investigated approach is the application of low-dose electromagnetic fields (EMF) to modulate cellular processes. A particular system is the BEMER therapy as a Physical Vascular Therapy for which a normalization of the microcirculation has been demonstrated by a low-frequency, pulsed EMF pattern. Open remains whether this EMF pattern impacts on cancer cell survival upon treatment with radiotherapy, chemotherapy and the molecular-targeted agent Cetuximab inhibiting the epidermal growth factor receptor. Using more physiological, three-dimensional, matrix-based cell culture models and cancer cell lines originating from lung, head and neck, colorectal and pancreas, we show significant changes in distinct intermediates of the glycolysis and tricarboxylic acid cycle pathways and enhanced cancer cell radiosensitization associated with increased DNA double strand break numbers and higher levels of reactive oxygen species upon BEMER treatment relative to controls. Intriguingly, exposure of cells to the BEMER EMF pattern failed to result in sensitization to chemotherapy and Cetuximab. Further studies are necessary to better understand the mechanisms underlying the cellular alterations induced by the BEMER EMF pattern and to clarify the application areas for human disease.

Sorafenib inhibits cell growth but fails to enhance radio- and chemosensitivity of glioblastoma cell lines.

BACKGROUND: Glioblastomas (GBM) are the most common malignant type of primary brain tumor. GBM are intensively treated with surgery and combined radiochemotherapy using X-irradiation and temozolomide (TMZ) but they are still associated with an extremely poor prognosis, urging for the development of new treatment strategies. To improve the outcome of GBM patients, the small molecule multi-kinase inhibitor sorafenib has moved into focus of recent research. Sorafenib has already been shown to enhance the radio- and radiochemosensitivity of other tumor entities. Whether sorafenib is also able to sensitize GBM cells to radio- and chemotherapy is still an unsolved question which we have addressed in this study. METHODS: The effect of sorafenib on signaling, proliferation, radiosensitivity, chemosensitivity and radiochemosensitivity was analyzed in six glioblastoma cell lines using Western blot, proliferation- and colony formation assays. RESULTS: In half of the cell lines sorafenib clearly inhibited MAPK signaling. We also observed a strong blockage of proliferation, which was, however, not associated with MAPK pathway inhibition. Sorafenib had only minor effects on cell survival when administered alone. Most importantly, sorafenib treatment failed to enhance GBM cell killing by irradiation, TMZ or combined treatment, and instead rather caused resistance in some cell lines. CONCLUSION: Our data suggest that sorafenib treatment may not improve the efficacy of radiochemotherapy in GBM.

TP53 mutant MDM2-amplified cell lines selected for resistance to MDM2-p53 binding antagonists retain sensitivity to ionizing radiation.

Non-genotoxic reactivation of the p53 pathway by MDM2-p53 binding antagonists is an attractive treatment strategy for wild-type TP53 cancers. To determine how resistance to MDM2/p53 binding antagonists might develop, SJSA-1 and NGP cells were exposed to growth inhibitory concentrations of chemically distinct MDM2 inhibitors, Nutlin-3 and MI-63, and clonal resistant cell lines generated. The p53 mediated responses of parental and resistant cell lines were compared. In contrast to the parental cell lines, p53 activation by Nutlin-3, MI-63 or ionizing radiation was not observed in either the SJSA-1 or the NGP derived cell lines. An identical TP53 mutation was subsequently identified in both of the SJSA-1 resistant lines, whilst one out of three identified mutations was common to both NGP derived lines. Mutation specific PCR revealed these mutations were present in parental SJSA-1 and NGP cell populations at a low frequency. Despite cross-resistance to a broad panel of MDM2/p53 binding antagonists, these MDM2-amplified and TP53 mutant cell lines remained sensitive to ionizing radiation (IR). These results indicate that MDM2/p53 binding antagonists will select for p53 mutations present in tumours at a low frequency at diagnosis, leading to resistance, but such tumours may nevertheless remain responsive to alternative therapies, including IR.

Hyperthermia adds to trabectedin effectiveness and thermal enhancement is associated with BRCA2 degradation and impairment of DNA homologous recombination repair.

The tetrahydroisoquinoline trabectedin is a marine compound with approved activity against human soft-tissue sarcoma. It exerts antiproliferative activity mainly by specific binding to the DNA and inducing DNA double-strand breaks (DSB). As homologous recombination repair (HRR)-deficient tumors are more susceptible to trabectedin, hyperthermia-mediated on-demand induction of HRR deficiency represents a novel and promising strategy to boost trabectedin treatment. For the first time, we demonstrate enhancement of trabectedin effectiveness in human sarcoma cell lines by heat and characterize cellular events and molecular mechanisms related to heat-induced effects. Hyperthermic temperatures (41.8 or 43°C) enhanced significantly trabectedin-related clonogenic cell death and G2/M cell cycle arrest followed by cell type-dependent induction of apoptosis or senescence. Heat combination increased accumulation of γH2AX foci as key marker of DSBs. Expression of BRCA2 protein, an integral protein of the HRR machinery, was significantly decreased by heat. Consequently, recruitment of downstream RAD51 to γH2AX-positive repair foci was almost abolished indicating relevant impairment of HRR by heat. Accordingly, enhancement of trabectedin effectiveness was significantly augmented in BRCA2-proficient cells by hyperthermia and alleviated in BRCA2 knockout or siRNA-transfected BRCA2 knockdown cells. In peripheral blood mononuclear cells isolated from sarcoma patients, increased numbers of nuclear γH2AX foci were detected after systemic treatment with trabectedin and hyperthermia of the tumor region. The findings establish BRCA2 degradation by heat as a key factor for a novel treatment strategy that allows targeted chemosensitization to trabectedin and other DNA damaging antitumor drugs by on-demand induction of HRR deficiency.

Magnetic fluid hyperthermia enhances cytotoxicity of bortezomib in sensitive and resistant cancer cell lines.

The proteasome inhibitor bortezomib (BZ) has shown promising results in some types of cancer, but in others it has had minimal activity. Recent studies have reported enhanced efficacy of BZ when combined with hyperthermia. However, the use of magnetic nanoparticles to induce hyperthermia in combination with BZ has not been reported. This novel hyperthermia modality has shown better potentiation of chemotherapeutics over other types of hyperthermia. We hypothesized that inducing hyperthermia via magnetic nanoparticles (MFH) would enhance the cytotoxicity of BZ in BZ-sensitive and BZ-resistant cancer cells more effectively than hyperthermia using a hot water bath (HWH). Studies were conducted using BZ in combination with MFH in two BZ-sensitive cell lines (MDA-MB-468, Caco-2), and one BZ-resistant cell line (A2780) at two different conditions, ie, 43°C for 30 minutes and 45°C for 30 minutes. These experiments were compared with combined application of HWH and BZ. The results indicate enhanced potentiation between hyperthermic treatment and BZ. MFH combined with BZ induced cytotoxicity in sensitive and resistant cell lines to a greater extent than HWH under the same treatment conditions. The observation that MFH sensitizes BZ-resistant cell lines makes this approach a potentially effective anticancer therapy platform.