Sestrin-3 modulation is essential for therapeutic efficacy of cucurbitacin B in lung cancer cells.

Many purified compounds from dietary sources have been investigated for their anticancer activities. The main issue with most agents is their effectiveness at high doses which generally could not be delivered to humans through dietary consumption. Here, we observed that cucurbitacin B, a tetracyclic triterpenoid present in pumpkins, gourds and squashes, exhibits antiproliferative effects on human non-small cell lung cancer (NSCLC) cells at nanomolar concentrations. Treatment with cucurbitacin B (0.2-0.6 µM; 24 h) was found to result in decrease in the viability of EGFR-wild type (A549 and H1792) and EGFR-mutant lung cancer cells (H1650 and H1975) and reduction in cell-colonies but had only minimal effect on normal human bronchial epithelial cells. Treatment with cucurbitacin B also caused inhibition of PI3K/mTOR and signal transducer and activator of transcription (STAT)-3 signaling along with simultaneous activation of AMPKα levels in both EGFR-wild type and EGFR-mutant lung cancer cells. Cucurbitacin B caused specific increase in the protein and mRNA expression of sestrin-3 in EGFR-mutant lung cancer cells, but not in EGFR-wild type cells. Treatment with cucurbitacin B to sestrin-3 siRNA treated EGFR-mutant cells further amplified the decrease in cell-viability and caused more sustained G2-phase cell cycle arrest, suggesting that these effects are mediated partly through sestrin-3. We also found that sestrin-3 has a role in the induction of apoptosis by cucurbitacin B in both EGFR-wild type and EGFR-mutant lung cancer cells. These findings suggest novel mechanism by the modulation of sestrin-3 for the action of cucurbitacin B and suggest that it could be developed as an agent for therapy of NSCLC.

AR-V7 exhibits non-canonical mechanisms of nuclear import and chromatin engagement in castrate-resistant prostate cancer.

Expression of the AR splice variant, androgen receptor variant 7 (AR-V7), in prostate cancer is correlated with poor patient survival and resistance to AR targeted therapies and taxanes. Currently, there is no specific inhibitor of AR-V7, while the molecular mechanisms regulating its biological function are not well elucidated. Here, we report that AR-V7 has unique biological features that functionally differentiate it from canonical AR-fl or from the second most prevalent variant, AR-v567. First, AR-V7 exhibits fast nuclear import kinetics via a pathway distinct from the nuclear localization signal dependent importin-α/ß pathway used by AR-fl and AR-v567. We also show that the dimerization box domain, known to mediate AR dimerization and transactivation, is required for AR-V7 nuclear import but not for AR-fl. Once in the nucleus, AR-V7 is transcriptionally active, yet exhibits unusually high intranuclear mobility and transient chromatin interactions, unlike the stable chromatin association of liganded AR-fl. The high intranuclear mobility of AR-V7 together with its high transcriptional output, suggest a Hit-and-Run mode of transcription. Our findings reveal unique mechanisms regulating AR-V7 activity, offering the opportunity to develop selective therapeutic interventions.

Quantitative analysis of taxane drug target engagement of microtubules in circulating tumor cells from metastatic castration resistant prostate cancer patients treated with CRXL301, a nanoparticle of docetaxel.

AIM: We reviewed the radiographic response of three patients with metastatic castration-resistant prostate cancer treated with CRXL301, a docetaxel nanoparticle. For these three patients, we isolated and analyzed circulating tumor cells (CTCs) to explore microtubule (MT) drug-target engagement (MT-DTE) as a biomarker of response to treatment. MT-DTE was based on a quantitative assessment of the MT cytoskeleton in CTCs from pre- and post-treatment patient samples as a potential read-out of CRXL301 activity. METHODS: We isolated CTCs using negative CD45+ depletion and subjected them to multiplex confocal microscopy using our established protocol. CTCs were identified as CD45-/CK+/DAPI+ cells and MT-DTE was determined using our developed imaging algorithm. We quantified MT bundling in CTCs across multiple time points, from baseline to on-treatment to disease progression. Here, we describe the longitudinal analysis of MT-DTE in CTCs from patients treated with CRXL301 and its correlation with response to treatment. RESULTS: We collected CTCs at seven time points from three metastatic castration-resistant prostate cancer patients. Clinical response was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) v.1.1 criteria in those patients with measurable disease. Of the three patients enrolled, one experienced partial response (-50%) to CRXL301 and two patients were unevaluable given bone only disease. Notably, however, these two patients showed stable disease clinically based on bone scans. MT-DTE across all time points revealed that, early time points within four and 24 h of drug administration exhibited the highest levels of drug engagement (MT-DTE) as compared to baseline. However, these early time points did not correlate with clinical response. We observed that the CTCs collected one week after the first or second dose of CRXL301 treatment in the responding patient had numerically higher levels of MT-DTE as compared to the other two patients. CONCLUSION: Taxane on-target activity can be detected and analyzed quantitatively in CTCs by tubulin immunofluorescence. Early time points, within 24 h of drug administration, showed high levels of DTE but did not correlate with clinical response. MT-DTE in CTCs collected after one week on treatment correlated best with treatment response. The clinical utility of the 1-week CTC DTE should be tested and validated in future clinical trials involving taxanes.

Fisetin Enhances Chemotherapeutic Effect of Cabazitaxel against Human Prostate Cancer Cells.

Although treatment of prostate cancer has improved over the past several years, taxanes, such as cabazitaxel, remain the only form of effective chemotherapy that improves survival in patients with metastatic castration-resistant prostate cancer. However, the effectiveness of this class of drugs has been associated with various side effects and drug resistance. We previously reported that fisetin, a hydroxyflavone, is a microtubule-stabilizing agent and inhibits prostate cancer cell proliferation, migration, and invasion and suggested its use as an adjuvant for treatment of prostate and other cancer types. In this study, we investigated the effect of fisetin in combination with cabazitaxel with the objective to achieve maximum therapeutic benefit, reduce dose and toxicity, and minimize or delay the induction of drug resistance and metastasis. Our data show for the first time that a combination of fisetin (20 µmol/L) enhances cabazitaxel (5 nmol/L) and synergistically reduces 22Rν1, PC-3M-luc-6, and C4-2 cell viability and metastatic properties with minimal adverse effects on normal prostate epithelial cells. In addition, the combination of fisetin with cabazitaxel was associated with inhibition of proliferation and enhancement of apoptosis. Furthermore, combination treatment resulted in the inhibition of tumor growth, invasion, and metastasis when assessed in two in vivo xenograft mouse models. These results provide evidence that fisetin may have therapeutic benefit for patients with advanced prostate cancer through enhancing the efficacy of cabazitaxel under both androgen-dependent and androgen-independent conditions. This study underscores the benefit of the combination of fisetin with cabazitaxel for the treatment of advanced and resistant prostate cancer and possibly other cancer types. Mol Cancer Ther; 15(12); 2863-74. ©2016 AACR.

Dietary flavonoid fisetin binds to ß-tubulin and disrupts microtubule dynamics in prostate cancer cells.

Microtubule targeting based therapies have revolutionized cancer treatment; however, resistance and side effects remain a major limitation. Therefore, novel strategies that can overcome these limitations are urgently needed. We made a novel discovery that fisetin, a hydroxyflavone, is a microtubule stabilizing agent. Fisetin binds to tubulin and stabilizes microtubules with binding characteristics far superior than paclitaxel. Surface plasmon resonance and computational docking studies suggested that fisetin binds to ß-tubulin with superior affinity compared to paclitaxel. Fisetin treatment of human prostate cancer cells resulted in robust up-regulation of microtubule associated proteins (MAP)-2 and -4. In addition, fisetin treated cells were enriched in α-tubulin acetylation, an indication of stabilization of microtubules. Fisetin significantly inhibited PCa cell proliferation, migration, and invasion. Nudc, a protein associated with microtubule motor dynein/dynactin complex that regulates microtubule dynamics, was inhibited with fisetin treatment. Further, fisetin treatment of a P-glycoprotein overexpressing multidrug-resistant cancer cell line NCI/ADR-RES inhibited the viability and colony formation. Our results offer in vitro proof-of-concept for fisetin as a microtubule targeting agent. We suggest that fisetin could be developed as an adjuvant for treatment of prostate and other cancer types.

Targeting microtubules by natural agents for cancer therapy.

Natural compounds that target microtubules and disrupt the normal function of the mitotic spindle have proven to be one of the best classes of cancer chemotherapeutic drugs available in clinics to date. There is increasing evidence showing that even minor alteration of microtubule dynamics can engage the spindle checkpoint, arresting cell-cycle progression at mitosis and subsequently leading to cell death. Our improved understanding of tumor biology and our continued appreciation for what the microtubule targeting agents (MTAs) can do have helped pave the way for a new era in the treatment of cancer. The effectiveness of these agents for cancer therapy has been impaired, however, by various side effects and drug resistance. Several new MTAs have shown potent activity against the proliferation of various cancer cells, including resistance to the existing MTAs. Sustained investigation of the mechanisms of action of MTAs, development and discovery of new drugs, and exploring new treatment strategies that reduce side effects and circumvent drug resistance could provide more effective therapeutic options for patients with cancer. This review focuses on the successful cancer chemotherapy from natural compounds in clinical settings and the challenges that may abort their usefulness.

Apoptosis and autophagy induction as mechanism of cancer prevention by naturally occurring dietary agents.

Nontoxic naturally occurring compounds, especially those from dietary sources, are receiving increasing consideration for prevention and treatment of diseases including cancer. There is a growing need for innovative anticancer therapies and therefore search for natural compounds with novel biological activities or antineoplastic potential is currently an important area in drug discovery. Support for this interest also comes from increasing concern over the efficacy and safety of many conventional therapies, especially those that run over a long course of time. Laboratory studies in different in vitro and in vivo systems have shown that many natural compounds possess the capacity to regulate response to oxidative stress and DNA damage, suppress angiogenesis, inhibit cell proliferation and induce autophagy and apoptosis. This review discusses the induction of apoptosis and autophagy as a mechanism of cancer prevention by some of the most studied naturally occurring dietary compounds.