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Evaluation of pharmacodynamic (PD) markers in vivo.

Nutlin-3a does not affect mutant p53 stability in TMD231 cells following cycloheximide treatment.

Single-agent or combination treatment does not affect TMD231 cell invasion.

Total apoptosis/necrosis in normal fibroblast cells.

Combination Nutlin-3a/carboplatin treatment decreases combination index (CI) values in a panel of breast cancer cell lines.

Supplemental Table S1. IC50 values for Nultin-3a, Carboplatin, and 1:1 Combination; Supplemental Material and Methods; Supplemental Figure Legends

Carboplatin and combination treatment induces accumulation in S and G2/M phases in TMD231 cells.

Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein–protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC. Mol Cancer Ther; 14(12); 2850–63. ©2015 AACR.

Telomeres serve the dual function of protecting chromosomes from genomic instability as well as protecting the ends of chromosomes from DNA damage machinery. The enzyme responsible for telomere maintenance is telomerase, an enzyme capable of reverse transcription. Telomerase activity is typically limited to specific cell types. However, telomerase activation in somatic cells serves as a key step toward cell immortalization and cancer. Targeting telomerase serves as a potential cancer treatment with significant therapeutic benefits. Beyond targeting cancers by inhibiting telomerase, manipulating the regulation of telomerase may also provide therapeutic benefit to other ailments, such as those related to aging. This review will introduce human telomeres and telomerase and discuss pharmacological regulation of telomerase, including telomerase inhibitors and activators, and their use in human diseases.

The use of botanical dietary supplements has grown steadily over the last 20 years despite incomplete information regarding active constituents, mechanisms of action, efficacy, and safety. An important but underinvestigated safety concern is the potential for popular botanical dietary supplements to interfere with the absorption, transport, and/or metabolism of pharmaceutical agents. Clinical trials of drug-botanical interactions are the gold standard and are usually carried out only when indicated by unexpected consumer side effects or, preferably, by predictive preclinical studies. For example, phase 1 clinical trials have confirmed preclinical studies and clinical case reports that St. John's wort (Hypericum perforatum) induces CYP3A4/CYP3A5. However, clinical studies of most botanicals that were predicted to interact with drugs have shown no clinically significant effects. For example, clinical trials did not substantiate preclinical predictions that milk thistle (Silybum marianum) would inhibit CYP1A2, CYP2C9, CYP2D6, CYP2E1, and/or CYP3A4. Here, we highlight discrepancies between preclinical and clinical data concerning drug-botanical interactions and critically evaluate why some preclinical models perform better than others in predicting the potential for drug-botanical interactions. Gaps in knowledge are also highlighted for the potential of some popular botanical dietary supplements to interact with therapeutic agents with respect to absorption, transport, and metabolism.

Resveratrol (RES) inhibits cell growth, induces apoptosis and augments chemotherapeutics in multiple cancer types, although its effects on drug-resistant cancer cells are unknown.To study the effects of resveratrol in triple-negative breast cancer cells that are resistant to the common cancer drug, paclitaxel, a novel paclitaxel-resistant cell line was generated from the MDA-MB-231 cell line.The resistant MDA-MB-231/PacR cells exhibited a 12-fold increased resistance to paclitaxel. RES treatment reduced cell proliferation and colony formation and increased senescence and apoptosis in both parental and resistant cells. Importantly, RES augmented the effects of paclitaxel in both cell lines. Up-regulation of the MDR1 and CYP2C8 genes were shown to be potential mechanisms of paclitaxel resistance in the resistant cells.RES, both alone and in combination with paclitaxel, may be useful in the treatment of paclitaxel-sensitive and paclitaxel-resistant triple-negative breast cancer cells.

Telomerase is a specialized reverse transcriptase that extends and maintains the terminal ends of chromosomes, or telomeres. Since its discovery in 1985 by Nobel Laureates Elizabeth Blackburn and Carol Greider, thousands of articles have emerged detailing its significance in telomere function and cell survival. This review provides a current assessment on the importance of telomerase regulation and relates it in terms of medical genetics. In this review, we discuss the recent findings on telomerase regulation, focusing on epigenetics and non-coding RNAs regulation of telomerase, such as microRNAs and the recently discovered telomeric-repeat containing RNA transcripts. Human genetic disorders that develop due to mutations in telomerase subunits, the role of single nucleotide polymorphisms in genes encoding telomerase components and diseases as a result of telomerase regulation going awry are also discussed. Continual investigation of the complex regulation of telomerase will further our insight into the use of controlling telomerase activity in medicine.

Treatment of drug-resistant cancer cells remains a difficult problem in cancer therapy because most resistant cells can pump out drugs or upregulate other survival pathways to bypass a targeted therapy. To study cancers that are resistant to the common cancer drug, paclitaxel, a novel paclitaxel-resistant cell line was generated from the breast cancer cell line MDA-MB-231. A “spiking” method of paclitaxel treatment was used to select for a population of cells that are resistant to the drug. This method mimics the development of resistance in recurrent tumors in patients. The IC50 of paclitaxel in these cells was 75 μM compared to the 0.037 μM IC50 of the parent cell line, a 2000-fold increase in resistance. In this very heterogeneous population, the mechanism of resistance was not due to increased protein levels of the efflux protein, p-glycoprotein, as quantitated by western blot analysis. To better study these cells, the paclitaxel-resistant cell line was cloned using a limiting dilution method to provide more homogeneous populations of resistant cells. The 29 clones obtained exhibited a paclitaxel IC50 range of 8 μM to 78 μM which was equivalent to a 200- to 2000-fold increase in resistance compared to the parent line. It has been suggested that the polyphenol natural compound, resveratrol, which has been shown to inhibit cell growth of multiple cancer types, may be useful as a combination anti-cancer treatment or novel therapeutic for drug-resistant cancer cells. There was no significant difference among the 72 hour IC50 of resveratrol in the parent line, the heterogeneous resistant line, the least paclitaxel-resistant clone or the most paclitaxel-resistant clone. We observed that treatment with 10-100 μM concentrations of resveratrol in all cell lines showed a reduction in cell proliferation and increased apoptosis within 72 hours (p Citation Format: Alyssa A. Sprouse, Brittney-Shea Herbert. Molecular mechanisms of paclitaxel-resistance and resveratrol sensitivity in MDA-MB-231 breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 876. doi:10.1158/1538-7445.AM2013-876

Treatment of drug-resistant cancer cells remains a difficult problem in cancer therapy because most resistant cells can pump out drugs or upregulate other survival pathways to bypass a targeted therapy. The polyphenol natural compound, resveratrol, has been shown to inhibit cell growth of multiple cancer types, but it is not cytotoxic to normal cells. However, the effects of resveratrol in triple negative breast cancer cells (estrogen receptor-, progesterone receptor-, and HER2-negative) as well as cancers that are resistant to the common cancer drug, paclitaxel, are not well understood. In this study, the effects of resveratrol were investigated in the triple negative breast cancer cell line MDA-MB-231 as well as a novel MDA-MB-231 derived paclitaxel-resistant line generated in our laboratory. Assays for cell proliferation, apoptosis, and protein expression changes related to apoptosis and survival were utilized in order to determine the differential effects of resveratrol on the parental and resistant cell lines. At lower (< 10 μM) concentrations, resveratrol induced cell proliferation in both cell lines, consistent with other studies demonstrating lifespan extension of normal cells with resveratrol treatment. On the other hand, after treatment with 10-100 μM concentrations of resveratrol, both cell lines exhibited a reduction in cell proliferation, with the paclitaxel-resistant cells to a greater extent. In addition, resveratrol decreased the ability of both cell lines to form colonies when plated at low density (an indication of reduced cell survival capacity). Furthermore, resveratrol treatment increased the amount of DNA fragmentation associated with apoptosis compared to untreated controls in both cell lines, but the paclitaxel resistant cells were more sensitive to resveratrol treatment than the parental cells. By protein expression analyses, we observed that in both the parental and paclitaxel-resistant cell lines, resveratrol may be acting through NAD-dependent deacetylase sirtuin (SIRT1) activity by decreasing the expression of the inhibitor-of-apoptosis protein, surviving and increasing the activator-of-apoptosis, caspase 3. Based on these data, we propose that resveratrol can inhibit proliferation and induce apoptosis in triple negative breast cancer cells, including paclitaxel-resistant cells. These results provide rationale for the use of resveratrol as an important starting point for the development of a novel anti-cancer agent for drug resistant, aggressive cancers as well as in combination with other anti-cancer drugs without significant toxicity to normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5676. doi:1538-7445.AM2012-5676