Natural Products Induce Lysosomal Membrane Permeabilization as an Anticancer Strategy.

Cancer is a global health and economic issue. The majority of anticancer therapies become ineffective due to frequent genomic turnover and chemoresistance. Furthermore, chemotherapy and radiation are non-specific, killing all rapidly dividing cells including healthy cells. In this review, we examine the ability of some natural products to induce lysosomal-mediated cell death in neoplastic cells as a way to kill them more specifically than conventional therapies. This list is by no means exhaustive. We postulate mechanisms to explain lysosomal membrane permeabilization and its role in triggering cell death in cancer cells.

Correction to: A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone.

The original article [1] contains three erroneous mentions of usage of a restriction enzyme-BstZ17I-in the Methods section as displayed in the following sentences.

Influence of lysosomal sequestration on multidrug resistance in cancer cells.

Chemotherapy remains a primary treatment modality for various malignancies. However, resistance to chemotherapeutic drugs is a major obstacle to curative cancer therapy. Lysosomes are acidic organelles that participate in cellular digestion. However, there is rising interest in lysosomes because of their involvement with cancer. For example, extracellular secretion of lysosomal enzymes promote tumorigenesis; cytosolic leakage of lysosomal hydrolases promote apoptosis; and weak chemotherapeutic bases diffuse across the lysosomal membrane and become entrapped in lysosomes in their cationic state. Lysosomal drug sequestration lowers the cytotoxic potential of chemotherapeutics, reduces drug availability to sites of action, and contributes to cancer resistance. This review examines various mechanisms of lysosomal drug sequestration and their consequences on cancer multidrug resistance. Strategies for overcoming drug resistance by exploiting lysosomes as subcellular targets to reverse drug sequestration and drug resistance are also discussed.

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone.

BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone. RESULTS: We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines. CONCLUSIONS: Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).

Dietary Isoflavones and Breast Cancer Risk.

Breast cancer is the deadliest neoplasm in women globally, resulting in a significant health burden. In many cases, breast cancer becomes resistant to chemotherapy, radiation, and hormonal therapies. It is believed that genetics is not the major cause of breast cancer. Other contributing risk factors include age at first childbirth, age at menarche, age at menopause, use of oral contraceptives, race and ethnicity, and diet. Diet has been shown to influence breast cancer incidence, recurrence, and prognosis. Soy isoflavones have long been a staple in Asian diets, and there appears to be an increase, albeit modest, compared to Asian populations, in soy consumption among Americans. Isoflavones are phytoestrogens that have antiestrogenic as well as estrogenic effects on breast cancer cells in culture, in animal models, and in clinical trials. This study will investigate anticancer and tumor promoting properties of dietary isoflavones and evaluate their effects on breast cancer development. Furthermore, this work seeks to elucidate the putative molecular pathways by which these phytochemicals modulate breast cancer risk by synergizing or antagonizing the estrogen receptor (ER) and in ER-independent signaling mechanisms.

Immunosuppressive, anti-inflammatory and anti-cancer properties of triptolide: A mini review.

OBJECTIVE: Triptolide, the active component of Tripterygium wilfordii Hook F has been used to treat autoimmune and inflammatory conditions for over two hundred years in traditional Chinese medicine. However, the processes through which triptolide exerts immunosuppression and anti-inflammation are not understood well. In this review, we discuss the autoimmune disorders and inflammatory conditions that are currently treated with triptolide. Triptolide also possesses anti-tumorigenic effects. We discuss the toxicity of various triptolide derivatives and offer suggestions to improve its safety. This study also examines the clinical trials that have investigated the efficacy of triptolide. Our aim is to examine the mechanisms that are responsible for the immunosuppressive, anti-inflammatory, and anti-cancer effects of triptolide. MATERIALS AND METHODS: The present review provides a comprehensive summary of the literature with respect to the immunosuppressive, anti-inflammatory, and anti-cancer properties of triptolide. RESULTS: Triptolide possesses immunosuppressive, anti-inflammatory, and anti-cancer effects. CONCLUSION: Triptolide can be used alone or in combination with existing therapeutic modalities as novel treatments for autoimmune disorders, cancers, and for immunosuppression.

Triptolide induces lysosomal-mediated programmed cell death in MCF-7 breast cancer cells.

BACKGROUND: Breast cancer is a major cause of death; in fact, it is the most common type, in order of the number of global deaths, of cancer in women worldwide. This research seeks to investigate how triptolide, an extract from the Chinese herb Tripterygium wilfordii Hook F, induces apoptosis in MCF-7 human breast cancer cells. Accumulating evidence suggests a role for lysosomal proteases in the activation of apoptosis. However, there is also some controversy regarding the direct participation of lysosomal proteases in activation of key apoptosis-related caspases and release of mitochondrial cytochrome c. In the present study, we demonstrate that triptolide induces an atypical, lysosomal-mediated apoptotic cell death in MCF-7 cells because they lack caspase-3. METHODS: MCF-7 cell death was characterized via cellular morphology, chromatin condensation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric cell growth inhibition assay and the expression levels of proapoptotic proteins. Acridine orange and LysoTracker® staining were performed to visualize lysosomes. Lysosomal enzymatic activity was monitored using an acid phosphatase assay and western blotting of cathepsin B protein levels in the cytosolic fraction, which showed increased enzymatic activity in drug-treated cells. RESULTS: These experiments suggest that triptolide-treated MCF-7 cells undergo atypical apoptosis and that, during the early stages, lysosomal enzymes leak into the cytosol, indicating lysosomal membrane permeability. CONCLUSION: Our results suggest that further studies are warranted to investigate triptolide's potential as an anticancer therapeutic agent.

Does apoptosis regulate the function of retinal photoreceptors?

Apoptosis, or programmed cell death, is an integral component of developmental biology, embryology, and anatomy. All eukaryotic cells possess the molecular machinery necessary to execute apoptosis. However, dysregulated apoptosis in the form of too much or too little cell death results in diseases such as Alzheimer's disease, autoimmune disorders, and cancer. It is postulated that apoptosis of the photoreceptors in the retina plays a vital role in mediating vision, and evidence is presented here to support this hypothesis. However, the precise mechanisms that regulate this cell death in photoreceptors have yet to be fully elucidated.