Proteasomal Degradation of Mcl-1 by Maritoclax Induces Apoptosis and Enhances the Efficacy of ABT-737 in Melanoma Cells

<div><p>Background and purpose</p><p>Metastatic melanoma remains one of the most invasive and highly drug resistant cancers. The over expression of anti-apoptotic protein Mcl-1 has been associated with inferior survival, poor prognosis and chemoresistance of malignant melanoma. A BH3 mimetic, ABT-737, has demonstrated efficacy in several forms of cancers. However, the efficacy of ABT-737 depends on Mcl-1. Because the over expression of Mcl-1 is frequently observed in melanoma, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells.</p><p>Experimental approach</p><p>For cell viability assessment we performed MTT assay. Apoptosis was determined using western blot and flow cytometric analysis.</p><p>Key results</p><p>The treatment of Maritoclax reduced the cell viability of melanoma cells with an IC₅₀ of between 2.2–5.0 µM. Further, treatment of melanoma cells with Maritoclax showed significant decrease in Mcl-1 expression. We found that Maritoclax was able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induced Mcl-1 degradation via the proteasome system, which was associated with its pro-apoptotic activity. We also found that Maritoclax treatment increased mitochondrial translocation of Bim and Bmf. Importantly, Maritoclax markedly enhanced the efficacy of ABT-737 against melanoma cells in both two- and three-dimensional spheroids.</p><p>Conclusions and implications</p><p>Taken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy for melanoma treatment that warrants further investigation as a single therapy or in combination with other agents such as Bcl-2 inhibitors.</p></div

Down regulation of Mcl-1 induces apoptosis in melanoma cells.

<p>Melanoma cells were incubated with either indicated amount of Maritoclax for 24h to access dose response or with 5 µM Maritoclax for mentioned time points. After incubation, cells were harvested; total lysates were prepared, and fractionated. The Western blot analysis was performed using mentioned antibodies and same blots were re-probed with either other antibodies or with anti-GAPDH to access loading. The results shown are representative of two independent experiments. <b><i>A-B,</i></b> Maritoclax had no effect on Bcl-2 and Bcl-xL. <b><i>C-D,</i></b> Degradation of Mcl-1 is associated with apoptosis.</p

Maritoclax sensitizes melanoma cells to ABT-737.

<p><b><i>A</i></b>, UACC903 cells were treated with increasing amount of ABT-737 (0.1–30 µM) alone (open circle) or together with 2.0 µM Maritoclax (closed circle) and then incubated for 24 h. Cell viabilities were determined by MTT assay. <b><i>B</i></b>, UACC903 cells were treated with either 5.0 µM of ABT-737 alone or together with 2.5 µM Maritoclax and then incubated for 24h. Cells were stained with a Live/Dead assay reagent for 30 min and then analyzed through flow cytometer. <b><i>C,</i></b> Effect of Maritoclax on apoptosis. UACC903 cells were treated with either 5.0 µM of ABT-737 alone or in combination with 2.5 µM Maritoclax and incubated for 24h. Cells were stained with Annexin-V-FITC and then analyzed through flow cytometer. <b><i>D-E,</i></b> Melanoma cells UACC903 were treated with 5.0 µM of ABT-737 and 2.5 µM of Maritoclax together for mentioned time points. After incubation, cells were harvested; total lysates were prepared, and fractionated. The Western blot analysis was performed using mentioned antibodies and same blots were re-probed with either other antibodies or with anti-GAPDH to access loading.</p

Design, Synthesis, and Biological Evaluation of Novel Selenium (Se-NSAID) Molecules as Anticancer Agents

The synthesis and anticancer evaluation of novel selenium-nonsteroidal anti-inflammatory drug (Se-NSAID) hybrid molecules are reported. The Se-aspirin analogue <b>8</b> was identified as the most effective agent in reducing the viability of different cancer cell lines, particularly colorectal cancer (CRC) cells, was more selective toward cancer cells than normal cells, and was >10 times more potent than 5-FU, the current therapy for CRC. Compound <b>8</b> inhibits CRC growth via the inhibition of the cell cycle in G1 and G2/M phases and reduces the cell cycle markers like cyclin E1 and B1 in a dose dependent manner; the inhibition of the cell cycle may be dependent on the ability of <b>8</b> to induce p21 expression. Furthermore, <b>8</b> induces apoptosis by activating caspase 3/7 and PARP cleavage, and its longer exposure causes increase in intracellular ROS levels in CRC cells. Taken together, <b>8</b> has the potential to be developed further as a chemotherapeutic agent for CRC

Design, Synthesis, and Biological Evaluation of Novel Selenium (Se-NSAID) Molecules as Anticancer Agents

The synthesis and anticancer evaluation of novel selenium-nonsteroidal anti-inflammatory drug (Se-NSAID) hybrid molecules are reported. The Se-aspirin analogue <b>8</b> was identified as the most effective agent in reducing the viability of different cancer cell lines, particularly colorectal cancer (CRC) cells, was more selective toward cancer cells than normal cells, and was >10 times more potent than 5-FU, the current therapy for CRC. Compound <b>8</b> inhibits CRC growth via the inhibition of the cell cycle in G1 and G2/M phases and reduces the cell cycle markers like cyclin E1 and B1 in a dose dependent manner; the inhibition of the cell cycle may be dependent on the ability of <b>8</b> to induce p21 expression. Furthermore, <b>8</b> induces apoptosis by activating caspase 3/7 and PARP cleavage, and its longer exposure causes increase in intracellular ROS levels in CRC cells. Taken together, <b>8</b> has the potential to be developed further as a chemotherapeutic agent for CRC

Insights into the Flexibility of ZIF‑7 and Its Structural Impact in Alcohol Adsorption

The flexibility of zeolitic imidazolate frameworks (ZIFs) is often interpreted as a result from their bulky linkers, the free rotations of which sometimes generate enough momentum to fluctuate the pore opening. In some extreme cases, all metal centers’ atomic positions are changed correspondingly, and in a three-dimensional network, it is intriguing how the displacement happens coordinately. Here, we bridged the learnings from zeolite sodalite (SOD) topology and applied it in studying ZIF-7’s structural transformation. We discovered a new extra-large pore ZIF-7 following the same rhombohedral symmetry. We have examined side-by-side the structural changes from X-ray diffraction (XRD) and solid-state NMR and successfully identified the precise adsorption sites. We applied ZIF-7’s flexibility in alcohol separation and used a series of alcohol molecules to study the adsorption mechanism

The Akt Inhibitor ISC-4 Synergizes with Cetuximab in 5-FU-Resistant Colon Cancer

<div><p>Phenylbutyl isoselenocyanate (ISC-4) is an Akt inhibitor with demonstrated preclinical efficacy against melanoma and colon cancer. In this study, we sought to improve the clinical utility of ISC-4 by identifying a synergistic combination with FDA-approved anti-cancer therapies, a relevant and appropriate disease setting for testing, and biomarkers of response. We tested the activity of ISC-4 and 19 FDA-approved anticancer agents, alone or in combination, against the SW480 and RKO human colon cancer cell lines. A synergistic interaction with cetuximab was identified and validated in a panel of additional colon cancer cell lines, as well as the kinetics of synergy. ISC-4 in combination with cetuximab synergistically reduced the viability of human colon cancer cells with wild-type but not mutant <i>KRAS</i> genes. Further analysis revealed that the combination therapy cooperatively decreased cell cycle progression, increased caspase-dependent apoptosis, and decreased phospho-Akt in responsive tumor cells. The synergism between ISC-4 and cetuximab was retained independently of acquired resistance to 5-FU in human colon cancer cells. The combination demonstrated synergistic anti-tumor effects <i>in vivo</i> without toxicity and in the face of resistance to 5-FU. These results suggest that combining ISC-4 and cetuximab should be explored in patients with 5-FU-resistant colon cancer harboring wild-type <i>KRAS</i>.</p> </div

ISC-4 and cetuximab cooperatively reduce phospho-Akt.

<p>(A) Western blot analysis of RKO cells treated with ISC-4 (2 µM) and cetuximab (1 µg/mL) alone or in combination for 24 hours. Densitometry of phospho-Akt levels are shown above the blot as normalized to Ran as relative to control treatment. (B) Western blot analysis of RKO cells treated with ISC-4 (2 µM) and cetuximab (1 µg/mL) alone or in combination for indicated time periods. Ran is shown as a loading control. (C) Western blot analysis of indicated human colon cancer cell lines following treatment with the combination (Rx) of ISC-4 (2 µM) and cetuximab (1 µg/mL) for 8 hrs. *<i>P</i><0.05 compared to control.</p

Effects of Cyr61 and Wnt5a on peripheral nerve regeneration.

<p>(A) Cyr61KD and MSC demonstrate no nerve regeneration at 4 weeks and poor nerve regeneration at 10 weeks. Wnt5aOX and MSC/CD34⁺ HSPC demonstrate increased early and robust nerve regeneration. Cyr61OX demonstrates nerve regeneration that continues to improve from 4 to 10 weeks. Data shown as means ± SEM (range). MSC and MSC/CD34⁺ HSPC data represent new quantitative assessment for a subset of samples from a previous study.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138643#pone.0138643.ref007" target="_blank">7</a>] (B) Sample photomicrographs demonstrate βIII tubulin⁽⁺⁾ (green) neuronal staining (rows 2 and 4, blue: DAPI, green arrows: regenerated nerves, white arrows: transition between native and regenerated tissue, R: regenerated tissue, N: native tissue). Masson’s trichrome-stained images are of a serial section of tissue for each sample (rows 1 and 3; black arrows: transition between native and regenerated tissue). Scale bar, 200 μm. (Unseeded, MSC and MSC/CD34⁺ HSPC images shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138643#pone.0138643.s002" target="_blank">S2 Fig</a>).</p

ISC-4 and cetuximab are cooperatively cytotoxic in 5-FU-resistant colon cancer cells.

<p>(A) Cell viability assays of RKO cells treated with ISC-4 (2 µM) and cetuximab (1 µg/mL) alone or in combination for the indicated time period (n = 3). (B) DAPI staining of RKO cells treated as in (A) for 12 hours. White arrows indicate cells with fragmented DNA. (C) Sub-G1 content of RKO cells treated with ISC-4 (2 µM) and cetuximab (1 µg/mL) alone or in combination for 12 hours (n = 3). *<i>P</i><0.05 compared to all treatment groups by Student's two-tailed <i>t</i> test. (D) Caspase-Glo assay of RKO cells treated with ISC-4 (2 uM) in combination with cetuximab (0, 0.25, 0.5, or 1 µg/mL) at 24 hours post-treatment. Bottom panel shows quantification of ISC-4 (2 uM) and cetuximab (1 µg/mL) (n = 3).</p